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

Previous studies have described the existence of high mobility group (HMG)-like proteins in macronuclei of the ciliated protozoan, Tetrahymena thermophila (Hamana, K., and K. Iwai, 1979, J. Biochem. [Tokyo], 69:1097-1111; Levy-Wilson, B., M. S. Denker, and E. Ito, 1983, Biochemistry, 22:1715-1721). In this report, two of these proteins, LG- 1 and LG-2, have been further characterized. Polyclonal antibodies raised against LG-1 and LG-2 fail to cross react with each other or any other macronuclear polypeptide in immunoblotting analyses. As well, LG- 1 and LG-2 antibodies do not react with calf thymus, chicken, or yeast HMG proteins. Consistent with these results, a 47 amino-terminal sequence of LG-1 has been determined that shows limited homology to both calf thymus HMGs 1 and 2 and HMGs 14 and 17. Two internal sequences of V8 protease-generated peptides from LG-2 have been determined, and these do not share any homology to the LG-1 sequence or any other sequenced HMG proteins. Comparison of the partial sequences of LG-1 and LG-2 with the complete amino acid sequence of the Tetrahymena histone H1 (Wu, M., C. D. Allis, R. Richman, R. G. Cook, and M. A. Gorovsky, 1986, Proc. Natl. Acad. Sci. USA, 83:8674-8678) rules out the possibility that LG-1 and LG-2 are proteolytically derived from H1, the other major macronuclear perchloric acid-soluble protein. Interestingly, however, both LG-1 and LG-2 are efficiently extracted from macronuclei by elutive intercalation (Schroter, H., G. Maier, H. Ponsting, and A. Nordheim, 1985, Embo (Eur. Mol. Biol. Organ.) J., 4:3867-3872), suggesting that both may share yet undetermined properties with HMGs 14 and 17 of higher eukaryotes. Examination of the pattern of LG-1 and LG-2 synthesis during the sexual phase of the life cycle, conjugation, demonstrates that the synthesis of LG-1 and LG-2 is coordinately increased from basal levels during the differentiation of new macronuclei (7-13 h), suggesting that both of these proteins play a role in determining a macronuclear phenotype. However, a specific induction of LG-2 synthesis is detected in early stages of conjugation (meiotic prophase, 1-4 h), leading to maximal synthesis of LG-2 at 3 h. Interestingly, the early induction of LG-2 synthesis closely parallels the hyperphosphorylation of histone H1. Taken together, these data suggest that LG-1 and LG-2 are not strongly related to each other or to higher eukaryotic HMG proteins.(ABSTRACT TRUNCATED AT 400 WORDS)     

Glycosylation, ADP-ribosylation, and methylation of Tetrahymena histones

We have examined some of the postsynthetic modifications that occur in macronuclear histones from Tetrahymena thermophila. When purified macronuclei are incubated with [32P]NAD+, histones H1, H2A, H2B, and H3 are ADP-ribosylated. Furthermore, histones H1, H2A, H2B, and H3 contain fucose and mannose residues as evidenced by the incorporation of [3H]fucose and by the specific binding to these proteins of gorse seed lectin and concanavalin A. Finally, our studies on incorporation of methyl groups into histones show that histone H2A, together with the related nonhistone protein A24, is methylated in Tetrahymena.     

Heterogeneous binding of high mobility group chromosomal proteins to nuclei

A dramatic difference is observed in the intracellular distribution of the high mobility group (HMG) proteins when chicken embryo fibroblasts are fractionated into nucleus and cytoplasm by either mass enucleation of cytochalasin-B-treated cells or by differential centrifugation of mechanically disrupted cells. Nuclei (karyoplasts) obtained by cytochalasin B treatment of cells contain more than 90 percent of the HMG 1, while enucleated cytoplasts contain the remainder. A similar distribution between karyoplasts and cytoplasts is observed for the H1 histones and the nucleosomal core histones as anticipated. The presence of these proteins, in low amounts, in the cytoplast preparation can be accounted for by the small percentage of unenucleated cells present. In contrast, the nuclei isolated from mechanically disrupted cells contain only 30-40 percent of the total HMGs 1 and 2, the remainder being recovered in the cytosol fraction. No histone is observed in the cytosol fraction. Unike the higher molecular weight HMGs, most of the HMGs 14 and 17 sediment with the nuclei after cell lysis by mechanical disruption. The distribution of HMGs is unaffected by incubating cells with cytochalasin B and mechanically fractionating rather than enucleating them. Therefore, the dramatic difference in HMG 1 distribution observed using the two fractionation techniques cannot be explained by a cytochalasin-B-induced redistribution. On reextraction and sedimentation of isolated nuclei obtained by mechanical cell disruption, only 8 percent of the HMG 1 is released to the supernate. Thus, the majority of the HMG 1 originally isolated with these nuclei, representing 35 percent of the total HMG 1, is stably bound, as is all the HMGs 14 and 17. The remaining 65 percent of the HMGs 1 and 2 is unstably bound and leaks to the cytosol fraction under the conditions of mechanical disruption. It is suggested that the unstably bound HMGs form a protein pool capable of equilibrating between cytoplasm and stably bound HMGs.     

Release of nucleosomes from nuclei by bleomycin-induced DNA strand scission

Mononucleosomes were released from both isolated mammalian (hog thyroid) and protozoan (Tetrahymena) nuclei by the bleomycin-induced DNA-strand breaking reaction. Trout sperm nuclei, on the other hand, were protected from the bleomycin-mediated DNA degradation. The mononucleosomes released from the bleomycin-treated nuclei contained the core histones H2A, H2B, H3, and H4; while HMG1 and HMG2 proteins, in addition to the core histones, were detected in the mononucleosomes obtained by micrococcal nuclease digestion of nuclei. HMGs, but not H1 histone, were dissociated into the supernatant by cleavage of chromatin DNA with bleomycin, whereas both HMGs and H1 were found in that fraction by digestion of nuclei with micrococcal nuclease. HMG1 and HMG2 were exclusively dissociated from chromatin with 1 mM bleomycin under the solvent condition where the DNA strand-breaking activity of the drug is repressed. These observations suggest the possibility that bleomycin preferentially binds to linker DNA regions not occupied by H1 histone in chromatin and exclusively dissociates HMG proteins and breaks the DNA strand. The results of the effects on bleomycin-induced DNA cleavage of nuclei of various drugs including polyamines, chelating agents, intercalating antibiotics such as mitomycin C or adriamycin, and radical scavengers are also presented.     

HMG-like chromosomal proteins in Trypanosoma cruzi.

HMG-like chromosomal proteins from Trypanosoma cruzi were studied. Four HMG-like proteins, designated HMG A, HMG-B, HMG-C, and HMG-E, were isolated and found to have molecular weights of 35.5 kd, 27.5 kd, 21.8 kd and 10.4 kd, respectively. Immunological relatedness was demonstrated between the mammalian HMG 1,2 and the HMG-A and HMG-B from T. cruzi. The relative amounts of HMG-C and HMG-E proteins vary in T. cruzi depending to the proliferative stage of the cells. HMG-E protein is increased in proliferating cells when compared to its level in non-proliferating cells. HMG-C is increased in the non-proliferating cells. Probably, the shifts observed in the relative amounts of HMG-like proteins are related to the proliferating cells of this flagellate. The results are consistent with those described for other lower eukaryotes where the HMG-like proteins isolated are similar but not identical to HMG proteins from vertebrates.     

High mobility group (HMG) proteins in the tammar wallaby Macropus eugenii: quantitative variations between tissues and testis-specific co-extracted proteins

1. Tammar wallaby (Macropus eugenii, Marsupialia) proteins with similar electrophoretic mobilities to calf non-histone chromosomal proteins HMG 1, 2, 14 and 17 are perchloric acid extracted from whole tissues (liver, kidney, spleen, brain and testis) and purified liver nuclei (using PCA or 0.35 M NaCl). 2. Tammar and calf HMG 1 have similar amino acid compositions. 3. Two testis-specific basic proteins co-extracting with HMG-like proteins from both tammar and red kangaroo (Megaleia rufa) are found in whole testis, purified testis nuclei, but not epididymis. 4. Tammar HMG 2 separates into two components on both acid urea and SDS gels. The larger, more basic protein, HMG 2b, is relatively abundant in proliferating tissues (testis, spleen).     

Tetrahymena HMG nonhistone chromosomal protein. Isolation and amino acid sequence lacking the N- and C-terminal domains of vertebrate HMG 1

The complete amino acid sequence of a high mobility group (HMG) nonhistone chromosomal protein of the ciliated protozoan Tetrahymena pyriformis (GL strain) was determined. This protein was extracted with 0.5 M HClO4 together with histone H1 (molar ratio 1:1) from the whole histone extract, then purified by gel filtration and reverse-phase HPLC. The HMG protein showed a single electrophoretic band on SDS gel electrophoresis. The amino acid sequence was determined by Edman degradation of intact protein, BrCN fragments, and their staphylococcal protease and tryptic peptides. Thus the total sequence, consisting of 99 amino acid residues and having a molecular weight of 11,626, was completely determined. Phosphorus analysis of the tryptic peptides, containing serine or threonine, showed that this HMG protein was phosphorylated at two positions, each 6-7%, and contained 0.15 mol phosphate/mol protein. This Tetrahymena HMG is rather similar to the central part of vertebrate HMG 1 in terms of the amino acid sequence and the hydropathy profile.     

Independent control during myogenesis of histone and high-mobility-group (HMGs) chromosomal protein heterogeneity

The ontogeny of high-mobility-group chromosomal protein and histone heterogeneity was followed in differentiating chicken skeletal muscle. The high-molecular-weight, high-mobility-group chromosomal proteins (HMGs 1, 2A, and 2B) showed a substantial change in their relative amounts during the period from 8 to 18 days. During this period HMG 2A decreases relative to HMG 1. There is also a decrease in the high-molecular-weight HMGs relative to the low-molecular-weight HMGs (HMGs 14 and 17). The microheterogeneity of the histones extracted from purified nuclei isolated from 8-, 11-, and 18-day embryonic myogenic tissue was analyzed by: (1) NaDodSO₄, (2) acid-urea, and (3) Triton acid-urea polyacrylamide gel electrophoresis. In contrast to the HMGs, the pattern of histone microheterogeneity for both H1 and H2A remained remarkably constant, despite the dramatic changes occurring in the rates of proliferation and the quality of gene expression in muscle cells during this developmental period. As would be expected from the decrease in cell proliferation occurring between days 8 and 18, increased amounts of phosphorylated H1 subspecies could be identified at the earlier stage. There was no change, however, in the proportion of the 4 major H1 parental variants. Thus, since the tissue-specific distribution of the H1s and HMGs are established at different times during myogenesis, it is likely that the two groups of chromatin proteins are involved in different events required for the establishment of the tissue-specific pattern of gene expression.     

Elimination of micronuclear specific DNA sequences early in anlagen development.

After conjugation in Tetrahymena thermophila, the old macronuclei degenerate, and new macronuclei (anlagen) develop. During anlagen development a number of DNA sequences found in the micronuclear genome (micronuclear limited sequences) are eliminated from the anlagen. A cloned copy of a repetitive micronuclear limited sequence has been used to determine the developmental stage at which micronuclear limited sequences are eliminated. DNAs from anlagen of various developmental stages were examined by Southern analysis. It was found that micronuclear limited sequences are present in 4C anlagen and essentially absent in 8C and 16C anlagen. The precipitous loss of these sequences in the 8C anlagen rules out under-replication as the mechanism for the loss and suggests that these sequences are specifically degraded early during anlagen development.