An intervening sequence in an unusual histone H1 gene of Tetrahymena thermophila.

Abstract

An intervening sequence of 254 base pairs interrupts the coding region ofthe single gene for macronuclear histone H1 of the ciliated protozoan, Tetrahymena thermophila. The intervening sequence has splice junctions similar to those found in RNA polymerase II genes of other organisms. No obvious similarities are observed between this intron and the self-splicing intervening sequence of the Tetrahymena ribosomal gene. The derived amino acid sequence describes a small extremely basic H1 protein missing most of the central hydrophobic domain that is conserved in all other H1 proteins. Macronuclei divide amitotically, without chromosome condensation, suggesting the conserved globular domain of H1 plays a role in higher-order chromatin structure.Histone H1 is located outside the nucleosome core. It has been implicated in determining the periodicity of nucleosome spacing (1-3), in completing the coiling of DNA around the nucleosome (4-14), in the higher-order structure of chromatin (4, 13-18), and in regulation of gene activity (19)(20)(21)(22)(23)(24) (60) of macronuclear DNA at low temperatures followed by sequential washes at higher temperature until autoradiographic analyses indicated hybridization to discrete bands. Prehybridization was in 5x SSPE (58), ix SPED (ix SPED is 6.0 mM NaDodSO4/20 mM sodium pyrophosphate/2 mM EDTA/0.1% Ficoll/0.1% polyvinylpyrrolidone 360/0.1% bovine serum albumin/5x Denhardt's solution (61) containing poly(A) (1 mg/ml) at 30°C overnight. Hybridization in fresh prehybridization solution containing probe at 106 cpm/ml was at 30°C for 5 hr. Filters were washed twice in 5 x SSPE for 15 min at 4°C, twice at room temperature, and finally at 47°C for probe 1 and at 50°C for probe 2. Each restriction enzyme gave a single band with both probes.Duplicate plaque lifts of a genomic library (49) were prepared (58) and hybridized separately with the two probes. Twelve of 60,000 plaques hybridized to both. All contained a 6.4-kilobase (kb) EcoRI insert that was subcloned into pUC13 to generate pTtDR1, which was mapped. A 2.4-kilobase-pair (kbp) EcoRI/HindIII fragment was subcloned into pUC18 to give pTtHi. A 1.1-kbp EcoRI/Taq I fragment from the clone was inserted into pUC18 to give pTR4. The EcoRI/Taq I fragment and 800-bp, 500-bp, and 180-bp Sau3A fragments of pTtH1 were subcloned into M13mpi8 and -mpi9 for sequencing.An additional mixed oligonucleotide was kindly provided by G. Childs. GANCCNGANGCNCC corresponds to the anti-sense of Gly-Ala-Ser-Gly-Ser, a sequence found in the central globular region of most H1 proteins. Stringent hybridization (62) failed to demonstrate any distinct bands in genomic blots of macronuclear DNA.DNA Sequence Analysis. DNA was sequenced (63) using a Bethesda Research Laboratories kit; the strategy is indicated Abbreviations: kbp, kilobase pair(s); pol II, RNA polymerase II. 8674The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solel...

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.

“…These probes were 5' end labeled with polynucleotide kinase and [y-32P]ATP (48) to a specific activity >108 cpm/4Lg and were purified by centrifugation through a Sephadex G-25 Quick Spin column (Boehringer Mannheim Biochemicals). Optimum hybridization conditions for all three probes were determined as described previously (62) and found to be 47°C for both hybridization and final wash. Each probe hybridized with a number of bands on genomic Southern blots. Some bands in a particular digest hybridized with more than one probe, suggesting that screening libraries for clones hybridizing to multiple probes would be a reasonable way to clone the gene.…”

“…Triplicate plaque lifts of a library containing partial EcoRIdigested macronuclear DNA fragments cloned in bacteriophage Xgt WES Xb (35) were prepared (48) and hybridized (62) separately with the three probes. A AgtlO cDNA library prepared from cells grown at 40°C (kindly provided by Gary Bannon) was also screened as described above.…”

“…Macronuclei contain an Hi-type histone whose solubility properties, extreme lysine richness, and cell growth/division-associated phosphorylation are typical of that class of histone. However, the derived (62) and actual (32) protein sequences of Tetrahymena macronuclear Hi indicate that it is unusual, inasmuch as it lacks some sequence elements and structural features associated with the conserved central globular core of Hi from higher organisms. Because Hi has been postulated to play an important role in higher-order chromatin structure (see reference 59 for a review), and macronuclei do not undergo mitotic chromosome condensation, it was suggested that the central globular core of Hi might be involved in-this process (62).…”

Tetrahymena contain two distinct and unusual high mobility group (HMG)-like proteins.

“…4 are not complete enough to rule out a possible precursor-product relationship between LG-2 and LG-1, it is worth noting that neither LG-1 or LG-2 can be derived from histone H1 as discussed previously. The complete amino acid sequence of the Tetrahymena histone H1 has recently been determined (Wu et al, 1986), and it does not contain any of the partial sequences of LG-1 or LG-2.…”

Chromatin from two classes of platyhelminthes display both protist H1 and higher eukaryote core histones

“…However, these chromosomal proteins present a high degree of divergence in lower eukaryotes, such as Entamoeba [1], Trichomonas [2], Trypanosoma [3][4][5][6][7][8], Leishmania and Crithidia [9], dinoflagellates [10], ciliata [11] and Giardia [12]. Histone divergence during evolution is expressed at the amino acid sequence level in core histones and mostly at the structural level in H1 histones.…”