The chromatin structure of a single cluster of six tandemly repeated 5S ribosomal RNA genes (5S genes) in Tetrahymena thermophila has been characterized. Indirect end labeling experiments indicate that the actively transcribed 5S genes in macronuclei are rapidly cut by DNAse I near the putative internal promotor and just 5' to the transcribed region. When cells are starved to reduce 5S gene transcription rates, the DNAse I sensitivity of the intragenic site is reduced relative to the 5' site. In the nontranscribed 5S genes in micronuclei, neither of these sites is hypersensitive to DNAse I. Thus structural alterations accompany both the activation of transcription during macronuclear development and physiological changes in the rate of transcription of the 5S genes. These DNAse I data together with studies using Staphylococcal nuclease suggest that rapidly transcribed 5S genes may not be associated with histones as nucleosomes. In contrast, the genes in starved cell macronuclei appear to be associated with one nucleosome per 280 base pair tandem repeat.
The relationship between chromatin structure and the transcriptional activity of the histone H4-I gene of Tetrahymena thermophila was explored. Indirect end-labeling studies demonstrated that major DNase I-and micrococcal nuclease-hypersensitive sites flank the active macronuclear genes but not the inactive micronuclear genes. Runon transcription experiments with isolated macronuclei indicated that histone gene transcription rates decreased when cells were starved. However, macronuclear nuclease-hypersensitive sites persisted upon starvation. Thus, one level of transcriptional control of the H4-I gene results in altered chromatin structure and is established during nuclear differentiation. The rate of transcription is also controlled, but not through hypersensitive site-associated structures.Because histone mRNA synthesis is often regulated both during differentiation and periodically through the cell cycle, histone genes provide a good model for studying the control of transcription. For this reason, we have been isolating and characterizing histone genes in Tetrahymena thermophila, a ciliated unicellular protozoan (3, 4). One of these genes, H4-I, codes for the major histone (H4) of T. thermophila. Like most or all genes in T. thermophila, it appears to be transcriptionally silent in the germinal micronucleus and active in the macronucleus. Frequent DNA sequence rearrangements (chromosome fragmentation, interstitial deletions) accompany gene activation during macronuclear development in T. thermophila (1,2,6,10,16,22,29,30). Although the role of these rearrangements, if any, in the expression of macronuclear genes has yet to be demonstrated (3, 22), they nonetheless offer the possibility that ciliates have unique mechanisms of gene activation. This possibility is heightened by reports that ciliates show peculiarities in the genetic code (11,14,15,23), suggesting that they are evolutionarily ancient. For the H4-I gene region, restriction maps for macro-and micronuclei are indistinguishable, indicating that no major sequence alterations accompany the activation of this gene during development.Unique sequence regions of the H4-I gene hybridize preferentially to one of two H4 cytoplasmic messages (both found on polysomes), indicating that it is not a pseudogene. It appears to be periodically active through the cell cycle (M. A. Gorovsky, unpublished observations) and, upon starvation, its abundance relative to other messages is diminished. This could reflect a shortened message half-life or a reduced rate of transcription during starvation. To distinguish between these possibilities, we labeled preinitiated transcripts in macronuclei from growing and starved cells by elongation in vitro. RNA from these reactions was hybridized to an excess of histone DNA on nitrocellulose filters, and the * Corresponding author. relative abundance of H4 gene transcripts was determined as previously described (21). The average rate of H4 message synthesis in seven separate determinations was about fourfold lower in starved than in ...
The relationship between chromatin structure and the transcriptional activity of the histone H4-I gene of Tetrahymena thermophila was explored. Indirect end-labeling studies demonstrated that major DNase I- and micrococcal nuclease-hypersensitive sites flank the active macronuclear genes but not the inactive micronuclear genes. Runon transcription experiments with isolated macronuclei indicated that histone gene transcription rates decreased when cells were starved. However, macronuclear nuclease-hypersensitive sites persisted upon starvation. Thus, one level of transcriptional control of the H4-I gene results in altered chromatin structure and is established during nuclear differentiation. The rate of transcription is also controlled, but not through hypersensitive site-associated structures.
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