Tetrahymena thermophila cells that had been shifted from log growth to a non-nutrient medium (60 mM Tris) were unable, during the first few hours of starvation, to mount a successful heat shock response and were killed by what should normally have been a nonlethal heat shock. An examination of the protein synthetic response of these short-starved cells during heat shock revealed that whereas they were able to initiate the synthesis of heat shock proteins, it was at a much reduced rate relative to controls and they quickly lost all capacity to synthesize any proteins. Certain pretreatments of cells, including a prior heat shock, abolished the heat shock inviability of these starved cells. Also, if cells were transferred to 10 mM Tris rather than 60 mM Tris, they were not killed by the same heat treatment. We found no abnormalities in either heat shock or non-heat shock mRNA metabolism in starved cells unable to survive a sublethal heat shock when compared with the response of those cells which can survive such a treatment. However, selective rRNA degradation occurred in the nonsurviving ceUs during the heat shock and this presumably accounted for their inviability. A prior heat shock administered to growing cells not only immunized them against the lethality of a heat shock while starved, but also prevented rRNA degradation from occurring.
The transcription of three specific genes has been examined in heat-shocked Drosophila cells by hybridizing pulse-labeled nuclear RNA with cloned DNA sequences. Actin gene transcription is rapidly and profoundly suppressed upon heat shock but returns to nearnormal levels after cells are placed back at their normal culture temperature (25°C) . Conversely, the transcription of genes coding for 70,000-and 26,000-dalton heat-shock proteins increases dramatically and with extraordinary rapidity (60 s) after heat shock. The temporal patterns of 70,000-and 26,000-dalton heat-shock gene transcription are nearly superimposable, indicating that, although they are not closely linked cytologically, these genes are nevertheless tightly coregulated. The abundance of heat-shock gene transcripts reaches remarkable levels, e.g ., 70,000-dalton heat-shock gene transcripts account for 2-3% of the nuclear RNA labeled during the first 30 min of heat shock. When heat-shocked cells are returned to 25°C, the rates of transcription of the heat-shock genes fall back to the low levels characteristic of untreated cells. To confirm the low level of heat-shock gene transcription in normal cells, nuclear RNA was purified from unlabeled (and otherwise unhandled) 25°C cells, end-labeled in vitro with 32 P, and hybridized to cloned heat-shock DNA sequences. These and other data establish that the genes for 70,000-and 26,000-dalton heat-shock proteins in cultured Drosophila cells are active at 25°C, and that their rate of transcription is greatly accelerated upon heat shock rather than being activated from a true "off" state. The rapidity, magnitude, and reversibility of the shifts in actin and heat-shock gene transcription constitute compelling advantages for the use of cultured Drosophila cells in studying the transcriptional regulation of eukaryotic genes, including one related to the cytoskeleton .A specific set of puffs is rapidly induced by heat shock in the polytene chromosomes of Drosophila larval salivary glands (25). This is accompanied by the synthesis of a small number of new polypeptides, the heat-shock proteins, and the cessation of most other translation in the cell (30). These initial observations have led to extensive studies of the chromosomal localization and DNA sequence organization of the genes directing the heat shock response, as well as analyses of messenger RNA and protein synthesis during heat shock (1) . It now appears that the Drosophila heat-shock response may be an example of a more general biological reaction to environmental stress because inducible genes coding for sets of proteins of molecular weights similar to those of the Drosophila heatshock proteins have been identified in a wide phyletic range of eukaryotes besides insects, including yeast, slime mold, and mammalian cells
Cells synthesize a characteristic set of proteins--heat shock proteins--in response to a rapid temperature jump or certain other stress treatments. The technique of phosphorus-31 nuclear magnetic resonance spectroscopy was used to examine in vivo the effects of temperature jump on two species of Tetrahymena that initiate the heat shock response at different temperatures. An immediate 50 percent decrease in cellular adenosine triphosphate was observed when either species was jumped to a temperature that strongly induces synthesis of heat shock proteins. This new adenosine triphosphate concentration was maintained at the heat shock temperature.
Tetrahymena thermophila cells that had been shifted from log growth to a non-nutrient medium (60 mM Tris) were unable, during the first few hours of starvation, to mount a successful heat shock response and were killed by what should normally have been a nonlethal heat shock. An examination of the protein synthetic response of these short-starved cells during heat shock revealed that whereas they were able to initiate the synthesis of heat shock proteins, it was at a much reduced rate relative to controls and they quickly lost all capacity to synthesize any proteins. Certain pretreatments of cells, including a prior heat shock, abolished the heat shock inviability of these starved cells. Also, if cells were transferred to 10 mM Tris rather than 60 mM Tris, they were not killed by the same heat treatment. We found no abnormalities in either heat shock or non-heat shock mRNA metabolism in starved cells unable to survive a sublethal heat shock when compared with the response of those cells which can survive such a treatment. However, selective rRNA degradation occurred in the nonsurviving cells during the heat shock and this presumably accounted for their inviability. A prior heat shock administered to growing cells not only immunized them against the lethality of a heat shock while starved, but also prevented rRNA degradation from occurring.
DNA in the polyploid macronucleus of the ciliated protozoan Tetrahymena thermophila contains the modified base N6-methyladenine. We identified two GATC sites which are methylated in most or all of the 45 copies of the macronuclear genome. One site is 2 kilobases 5' to the histone H4-I gene, and the other is 5 kilobases 3' to the 73-kilodalton heat shock protein gene. These sites are de novo methylated between 10 and 16 h after initiation of conjugation, during macronuclear anlage development. The methylation states of these two GATC sites and four other unmethylated GATC sites do not change in the DNA of cells cultured under conditions which change the activity of the genes, including logarithmic growth, starvation, and heat shock.The nuclear DNAs of most eucaryotes contain the modified base 5-methylcytosine (5MeC) as a minor component. In many cases, site-specific methylation of cytosine, particularly near the 5' ends of genes, has been correlated with gene inactivity (for reviews see references 1, 17, and 45). Therefore, it has been suggested that 5MeC may be involved in transcriptional regulation.N6-methyladenine (N6MeA) is found in the nuclear DNAs of several unicellular eucaryotes, either in combination with other modifications or as the only modified base. The DNAs of the green algae Chlamydomonas reinhardi and Chlorella spp. have both methylcytosine and methyladenine (28, 46), while only methylcytosine has been found in the DNA of another green alga, Euglena gracilis (40). In the ciliates Tetrahymena (23), Paramecium (16), Oxytricha (39), and Stylonychia (5) N6MeA is the sole modified base. The function of N6MeA in eucaryotic DNA is unknown.We examined methylation at specific sites in the genome of Tetrahymena thermophila. Tetrahymena cells contain two nuclei, a diploid micronucleus and a polyploid macronucleus. The macronucleus is responsible for most, if not all, transcriptional activity during vegetative growth. During sexual reproduction (conjugation), the macronucleus is destroyed, and mitotic products of the zygotic micronucleus develop into a new micronucleus and a new macronucleus. The developing macronucleus, called the anlage, undergoes many structural and morphological changes, including DNA replication to a final DNA content of 45c (49), elimination and rearrangement of germ line DNA sequences (50), and de novo methylation (9, 26).Although the micronuclear DNA of Tetrahymena cells does not contain modified bases, 0.8% of the adenine residues in the transcriptionally active macronucleus are modified to N6MeA (23,28). A partially purified methylase activity was isolated from macronuclei (10). A nearestneighbor analysis of in vivo methylated DNA showed that all four bases are found 5' to the methylated adenine, but only thymidine is found 3'. Some of the methyladenine residues in Tetrahymena macronuclear DNA occur in the sequence * Corresponding author. t Present address: Eleanor Roosevelt Cancer Institute, Denver, CO 80262.5'-GMeATC-3' and can be assayed by digestion with restriction enzyme DpnI...
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