A likely histone H2A.F/Z variant in Saccharomyces cerevisiae.
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Amino Acid Sequence↗
Biomedical subjects
M A Gorovsky
Publications and source records attributed to M A Gorovsky.
Either of the major H2A genes but not an evolutionarily conserved H2A.F/Z variant of Tetrahymena thermophila can function as the sole H2A gene in the yeast Saccharomyces cerevisiae.
H2A.F/Z histones are conserved variants that diverged from major H2A proteins early in evolution, suggesting they perform an important function distinct from major H2A proteins. Antisera specific for hv1, the H2A.F/Z variant of the ciliated protozoan Tetrahymena thermophila, cross-react with proteins from Saccharomyces cerevisiae. However, no H2A.F/Z variant has been reported in this budding yeast species. We sought to distinguish among three explanations for these observations: (i) that S. cerevisiae has an undiscovered H2A.F/Z variant, (ii) that the major S. cerevisiae H2A proteins are functionally equivalent to H2A.F/Z variants, or (iii) that the conserved epitope is found on a non-H2A molecule. Repeated attempts to clone an S. cerevisiae hv1 homolog only resulted in the cloning of the known H2A genes yHTA1 and yHTA2. To test for functional relatedness, we attempted to rescue strains lacking the yeast H2A genes with either the Tetrahymena major H2A genes (tHTA1 or tHTA2) or the gene (tHTA3) encoding hv1. Although they differ considerably in sequence from the yeast H2A genes, the major Tetrahymena H2A genes can provide the essential functions of H2A in yeast cells, the first such case of trans-species complementation of histone function. The Tetrahymena H2A genes confer a cold-sensitive phenotype. Although expressed at high levels and transported to the nucleus, hv1 cannot replace yeast H2A proteins. Proteins from S. cerevisiae strains lacking yeast H2A genes fail to cross-react with anti-hv1 antibodies. These studies make it likely that S. cerevisiae differs from most other eukaryotes in that it does not have an H2A.F/Z homolog. A hypothesis is presented relating the absence of H2A.F/Z in S. cerevisiae to its function in other organisms.
Amino Acid Sequence↗
Linker histones are not essential and affect chromatin condensation in vivo.
We have (separately) disrupted all of the expressed macronuclear copies of the HHO gene encoding macronuclear histone H1 and of the micronuclear linker histone (MLH) gene encoding the protein MicLH in Tetrahymena thermophila. These disruptions are shown to eliminate completely the expression of each protein. Strains without either linker histone grow at normal rates and reach near-normal cell densities, demonstrating that linker histones are not essential for cell survival. Histone H1 knockout (delta H1) cells have enlarged DAPI-stained macronuclei and normal-sized micronuclei, while MicLH knockout (delta MicLH) cells have enlarged micronuclei and normal-sized macronuclei. delta MicLH cells undergo mitosis normally. However, the micronuclear mitotic chromosome structure is less condensed. These studies provide evidence that linker histones are nonessential and are involved in chromatin packaging and condensation in vivo.
Animals↗
Requirement of a small cytoplasmic RNA for the establishment of thermotolerance.
Thermotolerance is an inducible state that endows cells with an enhanced resistance to thermal killing. Heat shock proteins are believed, and in a few instances have been shown, to be the agents conferring this resistance. The role of a small cytoplasmic RNA (G8 RNA) in developing thermotolerance in Tetrahymena thermophila was investigated by creating a strain devoid of all functional G8 genes. These G8 null cells mounted an apparently normal heat shock response, but they were unable to establish thermotolerance.
Adaptation, Physiological↗
DNA-mediated transformation in Tetrahymena.
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Animals↗
Strategies for the isolation of ciliary motility and assembly mutants in Tetrahymena.
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Animals↗
Acetylation of lysine 40 in alpha-tubulin is not essential in Tetrahymena thermophila.
In Tetrahymena, at least 17 distinct microtubule structures are assembled from a single primary sequence type of alpha- and beta-tubulin heterodimer, precluding distinctions among microtubular systems based on tubulin primary sequence isotypes. Tetrahymena tubulins also are modified by several types of posttranslational reactions including acetylation of alpha-tubulin at lysine 40, a modification found in most eukaryotes. In Tetrahymena, axonemal alpha-tubulin and numerous other microtubules are acetylated. We completely replaced the single type of alpha-tubulin gene in the macronucleus with a version encoding arginine instead of lysine 40 and therefore cannot be acetylated at this position. No acetylated tubulin was detectable in these transformants using a monoclonal antibody specific for acetylated lysine 40. Surprisingly, mutants lacking detectable acetylated tubulin are indistinguishable from wild-type cells. Thus, acetylation of alpha-tubulin at lysine 40 is non-essential in Tetrahymena. In addition, isoelectric focusing gel analysis of axonemal tubulin from cells unable to acetylate alpha-tubulin leads us to conclude that: (a) most or all ciliary alpha-tubulin is acetylated, (b) other lysines cannot be acetylated to compensate for loss of acetylation at lysine 40, and (c) acetylated alpha-tubulin molecules in wild-type cells contain one or more additional charge-altering modifications.
Acetylation↗
Gene-specific signal transduction between microtubules and tubulin genes in Tetrahymena thermophila.
Mammalian cells regulate tubulin mRNA abundance by a posttranscriptional mechanism dependent on the concentration of tubulin monomer. Treatment of mammalian cells with microtubule-depolymerizing drugs and microtubule-polymerizing drugs causes decreases and increases in tubulin mRNA, respectively (D. W. Cleveland, Curr. Opin. Cell Biol. 1:10-14, 1989). In striking contrast to the case with mammalian cells, perturbation of microtubules in Tetrahymena thermophila by microtubule-depolymerizing or -polymerizing drugs increases the level of the single alpha-tubulin gene message by increasing transcription (L. A. Stargell, D. P. Heruth, J. Gaertig, and M. A. Gorovsky, Mol. Cell. Biol. 12:1443-1450, 1992). In this report we show that antimicrotubule drugs preferentially induce the expression of one of two beta-tubulin genes (BTU1) in T. thermophila. In contrast, deciliation induces expression of both beta-tubulin genes. Tubulin gene expression was examined in a mutant strain created by transformation with an in vitro-mutagenized beta-tubulin gene that conferred resistance to microtubule-depolymerizing drugs and sensitivity to the polymerizing drug taxol and in a strain containing a nitrosoguanidine-induced mutation in the single alpha-tubulin gene that conferred the same pattern of drug sensitivities. In both cases the levels of tubulin mRNA expression from the drug-inducible BTU1 gene in the mutant cells paralleled the altered growth sensitivities to microtubule drugs. These studies demonstrate that T. thermophila has distinct, gene-specific mechanisms for modulating tubulin gene expression depending on whether ciliary or cytoplasmic microtubules are involved. They also show that the cytoplasmic microtubule cytoskeleton itself participates in a signal transduction pathway that regulates specific tubulin gene transcription in T. thermophila.
Animals↗
Gene transfer by electroporation in Tetrahymena.
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Animals↗
High frequency vector-mediated transformation and gene replacement in Tetrahymena.
Recently, we developed a mass DNA-mediated transformation technique for the ciliated protozoan Tetrahymena thermophila that introduces transforming DNA by electroporation into conjugating cells. Other studies demonstrated that a neomycin resistance gene flanked by Tetrahymena H4-I gene regulatory sequences transformed Tetrahymena by homologous recombination within the H4-I locus when microinjected into the macronucleus. We describe the use of conjugant electrotransformation (CET) for gene replacement and for the development of new independently replicating vectors and a gene cassette that can be used as a selectable marker in gene knockout experiments. Using CET, the neomycin resistance gene flanked by H4-I sequences transformed Tetrahymena, resulting in the replacement of the H4-I gene or integrative recombination of the H4-I/neo/H4-I gene (but not vector sequences) in the 5' or 3' flanking region of the H4-I locus. Gene replacement was obtained with non-digested plasmid DNA but releasing the insert increased the frequency of replacement events about 6-fold. The efficiency of transformation by the H4-I/neo/H4-I selectable marker was unchanged when a single copy of the Tetrahymena rDNA replication origin was included on the transforming plasmid. However, the efficiency of transformation using CET increased greatly when a tandem repeat of the replication origin fragment was used. This high frequency of transformation enabled mapping of the region required for H4-I promoter function to within 333 bp upstream of the initiator ATG. Similarly approximately 300 bp of sequence downstream of the translation terminator TGA of the beta-tubulin 2 (BTU2) gene could substitute for the 3' region of the H4-I gene. This hybrid H4-I/neo/BTU2 gene did not transform Tetrahymena when subcloned on a plasmid lacking an origin of replication, but did transform at high frequency on a two origin plasmid. Thus, the H4-I/neo/BTU2 cassette is a selectable marker that can be used for gene knockout in Tetrahymena. As a first step toward constructing a vector suitable for cloning genes by complementation of mutations in Tetrahymena, we also demonstrated that the vector containing 2 origins and the H4-I/neo/BTU2 cassette can co-express a gene encoding a cycloheximide resistant ribosomal protein.
Animals↗
Electroporation-mediated replacement of a positively and negatively selectable beta-tubulin gene in Tetrahymena thermophila.
Replacement of lysine-350 by methionine in the beta-tubulin gene of Chlamydomonas confers resistance to microtubule-depolymerizing drugs and increased sensitivity to the microtubule-stabilizing drug taxol. This mutation was created in cloned BTU1, one of two coexpressed beta-tublin genes of Tetrahymena thermophila. When introduced by electroporation, the mutated gene transformed Tetrahymena exclusively by gene replacement at the homologous locus. Taxol-sensitive transformants could be retransformed with a wild-type gene and selection for taxol resistance. Analyses of phenotypic assortment and of the mRNA in transformed cells suggest that complete replacement of the BTU1 gene in the polyploid macronucleus can be obtained. These studies demonstrate the utility of this marker for studying tublin gene function and show that electroporation allows facile gene replacement in Tetrahymena.
Animals↗
Phylogenetic analysis of the core histones H2A, H2B, H3, and H4.
Despite the ubiquity of histones in eukaryotes and their important role in determining the structure and function of chromatin, no detailed studies of the evolution of the histones have been reported. We have constructed phylogenetic trees for the core histones H2A, H2B, H3, and H4. Histones which form dimers (H2A/H2B and H3/H4) have very similar trees and appear to have co-evolved, with the exception of the divergent sea urchin testis H2Bs, for which no corresponding divergent H2As have been identified. The trees for H2A and H2B also support the theory that animals and fungi have a common ancestor. H3 and H4 are 10-fold less divergent than H2A and H2B. Three evolutionary histories are observed for histone variants. H2A.F/Z-type variants arose once early in evolution, while H2A.X variants arose separately, during the evolution of multicellular animals. H3.3-type variants have arisen in multiple independent events.
Animals↗
Independent evolutionary origin of histone H3.3-like variants of animals and Tetrahymena.
All three genes encoding histone H3 proteins were cloned and sequenced from Tetrahymena thermophila. Two of these genes encode a major H3 protein identical to that of T. pyriformis and 87% identical to the major H3 of vertebrates. The third gene encodes hv2, a quantitatively minor replication independent (replacement) variant. The sequence of hv2 is only 85% identical to the animal replacement variant H3.3 and is the most divergent H3 replacement variant described. Phylogenetic analysis of 73 H3 protein sequences suggests that hv2, H3.3, and the plant replacement variant H3.III evolved independently, and that H3.3 is not the ancestral H3 gene, as was previously suggested (Wells, D., Bains, W., and Kedes, L. 1986, J. Mol. Evol., 23: 224-241). These results suggest it is the replication independence and not the particular protein sequence that is important in the function of H3 replacement variants.
Amino Acid Sequence↗
Regulation and evolution of the single alpha-tubulin gene of the ciliate Tetrahymena thermophila.
The single alpha-tubulin gene of Tetrahymena thermophila was isolated from a genomic library and shown to encode a single protein. Comparisons of the rates of evolution of this gene with other alpha-tubulin sequences revealed that it belongs to a group of more evolutionarily constrained alpha-tubulin proteins in animals, plants, and protozoans versus the group of more rapidly evolving fungal and variant animal alpha-tubulins. The single alpha-tubulin of Tetrahymena must be used in a variety of microtubule structures, and we suggest that equivalently conserved alpha-tubulins in other organisms are evolutionarily constrained because they, too, are multifunctional. Reduced constraints on fungal tubulins are consistent with their simpler microtubule systems. The animal variant alpha-tubulins may also have diverged because of fewer functional requirements or they could be examples of specialized tubulins. To analyze the role of tubulin gene expression in regulation of the complex microtubule system of Tetrahymena, alpha-tubulin mRNA amounts were examined in a number of cell states. Message levels increased in growing versus starved cells and also during early stages of conjugation. These changes were correlated with increases in transcription rates. Additionally, alpha-tubulin mRNA levels oscillate in a cell cycle dependent fashion caused by changes in both transcription and decay rates. Therefore, as in other organisms, Tetrahymena adjusts alpha-tubulin message amounts via message decay. However the complex control of alpha-tubulin mRNA during the Tetrahymena life cycle involves regulation of both decay and transcription rates.
Amino Acid Sequence↗
Four distinct and unusual linker proteins in a mitotically dividing nucleus are derived from a 71-kilodalton polyprotein, lack p34cdc2 sites, and contain protein kinase A sites.
Tetrahymena thermophila micronuclei contain four linker-associated proteins, alpha, beta, gamma, and delta. Synthetic oligonucleotides based on N-terminal protein sequences of beta and gamma were used to clone the micronuclear linker histone (MLH) gene. The MLH gene is single copy and is transcribed into a 2.4-kb message encoding all four linker-associated proteins. The message is translated into a polypeptide (Mic LH) that is processed at the sequence decreases RTK to give proteins whose amino acid sequences differ markedly from each other, from the sequence of macronuclear H1, and from sequences of typical H1s of other organisms. This represents the first example of multiple chromatin proteins derived from a single polyprotein. The delta protein consists largely of two high-mobility-group (HMG) boxes. An evolutionary analysis of HMG boxes indicates that the delta HMG boxes are similar to the HMG boxes of tsHMG, a protein that appears in elongating mouse spermatids when they condense and cease transcription, suggesting that delta could play a similar role in the micronucleus. The micronucleus divides mitotically, while the macronucleus divides amitotically. Surprisingly, macronuclear H1 but not Mic LH contains sequences resembling p34cdc2 kinase phosphorylation sites, while each of the Mic LH-derived proteins contains a typical protein kinase A phosphorylation site in its carboxy terminus.
Amino Acid Sequence↗
TATA-binding protein and nuclear differentiation in Tetrahymena thermophila.
Unambiguous TATA boxes have not been identified in upstream sequences of Tetrahymena thermophila genes analyzed to date. To begin a characterization of the promoter requirements for RNA polymerase II, the gene encoding TATA-binding protein (TBP) was cloned from this species. The derived amino acid sequence for the conserved C-terminal domain of Tetrahymena TBP is one of the most divergent described and includes a unique 20-amino-acid C-terminal extension. Polyclonal antibodies generated against a fragment of Tetrahymena TBP recognize a 36-kDa protein in macronuclear preparations and also cross-react with yeast and human TBPs. Immunocytochemistry was used to examine the nuclear localization of TBP during growth, starvation, and conjugation (the sexual phase of the life cycle). The transcriptionally active macronuclei stained at all stages of the life cycle. The transcriptionally inert micronuclei did not stain during growth or starvation but surprisingly stained with anti-TBP throughout early stages of conjugation. Anti-TBP staining disappeared from developing micronuclei late in conjugation, corresponding to the onset of transcription in developing macronuclei. Since micronuclei do not enlarge or divide at this time, loss of TBP appears to be an active process. Thus, the transcriptional differences between macro- and micronuclei that arise during conjugation are associated with the loss of a major component of the basal transcription apparatus from developing micronuclei rather than its appearance in developing macronuclei.
Amino Acid Sequence↗
Mapping the 5' and 3' ends of Tetrahymena thermophila mRNAs using RNA ligase mediated amplification of cDNA ends (RLM-RACE).
A procedure is described for mapping the ends of RNAs. Using T4 RNA ligase, a DNA (3' end) or RNA (5' end) oligonucleotide is ligated to RNA ends followed by cDNA synthesis, PCR amplification, cloning and sequencing. This method determines 5' ends, 3' polyadenylation sites and the size of poly(A) tails, and should be applicable to non-polyadenylated mRNAs and to non-message RNAs. Analysis of four Tetrahymena thermophila histone mRNAs revealed multiple, closely spaced 5' ends consistent with those determined by other methods. Except for a 'CCAAT' box in either orientation 100-200 nucleotides upstream of the transcription start site, no conserved sequence elements were observed in the untranslated 5' region or in sequences immediately flanking the transcription start site. Analysis of the 3' ends of mRNAs encoding four histones, two tubulins and the Tetrahymena TATA binding protein confirmed the observations that Tetrahymena histone messages are polyadenylated and that poly(A) tails in this organism are short (approximately 50 nt). No canonical poly(A) addition signal was identified. The four histone messages analyzed have contained three sequence elements, TGTGT-TAA-AAGTATT, not found in non-histone messages. Two non-histone messages contained GCATT(N)15ATACC near the poly(A) addition site.
Animals↗
Perspectives on tubulin isotype function and evolution based on the observation that Tetrahymena thermophila microtubules contain a single alpha- and beta-tubulin.
We have cloned and sequenced the two beta-tubulin genes of the ciliated protozoan Tetrahymena thermophila. The two genes encode identical 443 amino acid peptides which are 99.7% identical to the beta-tubulin proteins of T. pyriformis and 95% identical to human beta 1 tubulin. T. thermophila contains only one alpha-tubulin gene (Callahan et al., 1984: Cell 36:441-445). Thus, all of the extremely diverse microtubule structures in this unicellular organism can be formed from a single alpha- and a single beta-tubulin peptide. We have also carried out a phylogenetic analysis of 84 complete beta-tubulin peptide sequences. This analysis supports two hypotheses regarding beta-tubulin evolution and function: 1) Multifunctional beta-tubulins are under greater evolutionary constraint than beta-tubulins present in specialized cells or in cells with very few microtubule related functions, which can evolve rapidly; and 2) Cells which form axonemes maintain a homogeneous population of tubulins.
Amino Acid Sequence↗