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. 1994 Jan;14(1):10–20. doi: 10.1128/mcb.14.1.10

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.

M Wu 1, C D Allis 1, M T Sweet 1, R G Cook 1, T H Thatcher 1, M A Gorovsky 1
PMCID: PMC358351  PMID: 8264578

Abstract

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.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Adam S. A., Gerace L. Cytosolic proteins that specifically bind nuclear location signals are receptors for nuclear import. Cell. 1991 Sep 6;66(5):837–847. doi: 10.1016/0092-8674(91)90431-w. [DOI] [PubMed] [Google Scholar]
  2. Allis C. D., Allen R. L., Wiggins J. C., Chicoine L. G., Richman R. Proteolytic processing of h1-like histones in chromatin: a physiologically and developmentally regulated event in Tetrahymena micronuclei. J Cell Biol. 1984 Nov;99(5):1669–1677. doi: 10.1083/jcb.99.5.1669. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Allis C. D., Glover C. V., Bowen J. K., Gorovsky M. A. Histone variants specific to the transcriptionally active, amitotically dividing macronucleus of the unicellular eucaryote, Tetrahymena thermophila. Cell. 1980 Jul;20(3):609–617. doi: 10.1016/0092-8674(80)90307-4. [DOI] [PubMed] [Google Scholar]
  4. Allis C. D., Glover C. V., Gorovsky M. A. Micronuclei of Tetrahymena contain two types of histone H3. Proc Natl Acad Sci U S A. 1979 Oct;76(10):4857–4861. doi: 10.1073/pnas.76.10.4857. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Allis C. D., Gorovsky M. A. Histone phosphorylation in macro- and micronuclei of Tetrahymena thermophila. Biochemistry. 1981 Jun 23;20(13):3828–3833. doi: 10.1021/bi00516a025. [DOI] [PubMed] [Google Scholar]
  6. Allis C. D., Richman R., Gorovsky M. A., Ziegler Y. S., Touchstone B., Bradley W. A., Cook R. G. hv1 is an evolutionarily conserved H2A variant that is preferentially associated with active genes. J Biol Chem. 1986 Feb 5;261(4):1941–1948. [PubMed] [Google Scholar]
  7. Bannon G. A., Calzone F. J., Bowen J. K., Allis C. D., Gorovsky M. A. Multiple, independently regulated, polyadenylated messages for histone H3 and H4 in Tetrahymena. Nucleic Acids Res. 1983 Jun 25;11(12):3903–3917. doi: 10.1093/nar/11.12.3903. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Bianchi M. E., Falciola L., Ferrari S., Lilley D. M. The DNA binding site of HMG1 protein is composed of two similar segments (HMG boxes), both of which have counterparts in other eukaryotic regulatory proteins. EMBO J. 1992 Mar;11(3):1055–1063. doi: 10.1002/j.1460-2075.1992.tb05144.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Boissonneault G., Lau Y. F. A testis-specific gene encoding a nuclear high-mobility-group box protein located in elongating spermatids. Mol Cell Biol. 1993 Jul;13(7):4323–4330. doi: 10.1128/mcb.13.7.4323. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Bruhn S. L., Housman D. E., Lippard S. J. Isolation and characterization of cDNA clones encoding the Drosophila homolog of the HMG-box SSRP family that recognizes specific DNA structures. Nucleic Acids Res. 1993 Apr 11;21(7):1643–1646. doi: 10.1093/nar/21.7.1643. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Brunk C. F., Sadler L. A. Characterization of the promoter region of Tetrahymena genes. Nucleic Acids Res. 1990 Jan 25;18(2):323–329. doi: 10.1093/nar/18.2.323. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Cedergren R., Gray M. W., Abel Y., Sankoff D. The evolutionary relationships among known life forms. J Mol Evol. 1988 Dec;28(1-2):98–112. doi: 10.1007/BF02143501. [DOI] [PubMed] [Google Scholar]
  13. Chan R. L., Keller M., Canaday J., Weil J. H., Imbault P. Eight small subunits of Euglena ribulose 1-5 bisphosphate carboxylase/oxygenase are translated from a large mRNA as a polyprotein. EMBO J. 1990 Feb;9(2):333–338. doi: 10.1002/j.1460-2075.1990.tb08115.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Chicoine L. G., Wenkert D., Richman R., Wiggins J. C., Allis C. D. Modulation of linker histones during development in Tetrahymena: selective elimination of linker histone during the differentiation of new macronuclei. Dev Biol. 1985 May;109(1):1–8. doi: 10.1016/0012-1606(85)90339-2. [DOI] [PubMed] [Google Scholar]
  15. Dadd C. A., Cook R. G., Allis C. D. Fractionation of small tryptic phosphopeptides by alkaline PAGE followed by amino acid sequencing. Biotechniques. 1993 Feb;14(2):266–273. [PubMed] [Google Scholar]
  16. Devereux J., Haeberli P., Smithies O. A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res. 1984 Jan 11;12(1 Pt 1):387–395. doi: 10.1093/nar/12.1part1.387. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Douglass J., Civelli O., Herbert E. Polyprotein gene expression: generation of diversity of neuroendocrine peptides. Annu Rev Biochem. 1984;53:665–715. doi: 10.1146/annurev.bi.53.070184.003313. [DOI] [PubMed] [Google Scholar]
  18. Feinberg A. P., Vogelstein B. A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal Biochem. 1983 Jul 1;132(1):6–13. doi: 10.1016/0003-2697(83)90418-9. [DOI] [PubMed] [Google Scholar]
  19. Ferrari S., Harley V. R., Pontiggia A., Goodfellow P. N., Lovell-Badge R., Bianchi M. E. SRY, like HMG1, recognizes sharp angles in DNA. EMBO J. 1992 Dec;11(12):4497–4506. doi: 10.1002/j.1460-2075.1992.tb05551.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Gething M. J., Sambrook J. Protein folding in the cell. Nature. 1992 Jan 2;355(6355):33–45. doi: 10.1038/355033a0. [DOI] [PubMed] [Google Scholar]
  21. Gorovsky M. A. Genome organization and reorganization in Tetrahymena. Annu Rev Genet. 1980;14:203–239. doi: 10.1146/annurev.ge.14.120180.001223. [DOI] [PubMed] [Google Scholar]
  22. Gorovsky M. A., Keevert J. B., Pleger G. L. Histone F1 of Tetrahymena macronuclei: unique electrophoretic properties and phosphorylation of F1 in an amitotic nucleus. J Cell Biol. 1974 Apr;61(1):134–145. doi: 10.1083/jcb.61.1.134. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Gorovsky M. A. Macro- and micronuclei of Tetrahymena pyriformis: a model system for studying the structure and function of eukaryotic nuclei. J Protozool. 1973 Feb;20(1):19–25. doi: 10.1111/j.1550-7408.1973.tb05995.x. [DOI] [PubMed] [Google Scholar]
  24. Gorovsky M. A., Yao M. C., Keevert J. B., Pleger G. L. Isolation of micro- and macronuclei of Tetrahymena pyriformis. Methods Cell Biol. 1975;9(0):311–327. doi: 10.1016/s0091-679x(08)60080-1. [DOI] [PubMed] [Google Scholar]
  25. Griess E. A., Rensing S. A., Grasser K. D., Maier U. G., Feix G. Phylogenetic relationships of HMG box DNA-binding domains. J Mol Evol. 1993 Aug;37(2):204–210. doi: 10.1007/BF02407357. [DOI] [PubMed] [Google Scholar]
  26. Hammarback J. A., Obar R. A., Hughes S. M., Vallee R. B. MAP1B is encoded as a polyprotein that is processed to form a complex N-terminal microtubule-binding domain. Neuron. 1991 Jul;7(1):129–139. doi: 10.1016/0896-6273(91)90081-a. [DOI] [PubMed] [Google Scholar]
  27. Hayashi T., Hayashi H., Iwai K. Tetrahymena histone H1. Isolation and amino acid sequence lacking the central hydrophobic domain conserved in other H1 histones. J Biochem. 1987 Aug;102(2):369–376. doi: 10.1093/oxfordjournals.jbchem.a122063. [DOI] [PubMed] [Google Scholar]
  28. Hellen C. U., Kräusslich H. G., Wimmer E. Proteolytic processing of polyproteins in the replication of RNA viruses. Biochemistry. 1989 Dec 26;28(26):9881–9890. doi: 10.1021/bi00452a001. [DOI] [PubMed] [Google Scholar]
  29. Hirata R., Ohsumk Y., Nakano A., Kawasaki H., Suzuki K., Anraku Y. Molecular structure of a gene, VMA1, encoding the catalytic subunit of H(+)-translocating adenosine triphosphatase from vacuolar membranes of Saccharomyces cerevisiae. J Biol Chem. 1990 Apr 25;265(12):6726–6733. [PubMed] [Google Scholar]
  30. Horowitz S., Bowen J. K., Bannon G. A., Gorovsky M. A. Unusual features of transcribed and translated regions of the histone H4 gene family of Tetrahymena thermophila. Nucleic Acids Res. 1987 Jan 12;15(1):141–160. doi: 10.1093/nar/15.1.141. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Hsiao K. C., Cheng C. H., Fernandes I. E., Detrich H. W., DeVries A. L. An antifreeze glycopeptide gene from the antarctic cod Notothenia coriiceps neglecta encodes a polyprotein of high peptide copy number. Proc Natl Acad Sci U S A. 1990 Dec;87(23):9265–9269. doi: 10.1073/pnas.87.23.9265. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Hunkapiller M. W., Lujan E., Ostrander F., Hood L. E. Isolation of microgram quantities of proteins from polyacrylamide gels for amino acid sequence analysis. Methods Enzymol. 1983;91:227–236. doi: 10.1016/s0076-6879(83)91019-4. [DOI] [PubMed] [Google Scholar]
  33. Jantzen H. M., Admon A., Bell S. P., Tjian R. Nucleolar transcription factor hUBF contains a DNA-binding motif with homology to HMG proteins. Nature. 1990 Apr 26;344(6269):830–836. doi: 10.1038/344830a0. [DOI] [PubMed] [Google Scholar]
  34. Kane P. M., Yamashiro C. T., Wolczyk D. F., Neff N., Goebl M., Stevens T. H. Protein splicing converts the yeast TFP1 gene product to the 69-kD subunit of the vacuolar H(+)-adenosine triphosphatase. Science. 1990 Nov 2;250(4981):651–657. doi: 10.1126/science.2146742. [DOI] [PubMed] [Google Scholar]
  35. Kennelly P. J., Krebs E. G. Consensus sequences as substrate specificity determinants for protein kinases and protein phosphatases. J Biol Chem. 1991 Aug 25;266(24):15555–15558. [PubMed] [Google Scholar]
  36. Kolodrubetz D. Consensus sequence for HMG1-like DNA binding domains. Nucleic Acids Res. 1990 Sep 25;18(18):5565–5565. doi: 10.1093/nar/18.18.5565. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Lamb N. J., Cavadore J. C., Labbe J. C., Maurer R. A., Fernandez A. Inhibition of cAMP-dependent protein kinase plays a key role in the induction of mitosis and nuclear envelope breakdown in mammalian cells. EMBO J. 1991 Jun;10(6):1523–1533. doi: 10.1002/j.1460-2075.1991.tb07672.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Landsman D., Bustin M. A signature for the HMG-1 box DNA-binding proteins. Bioessays. 1993 Aug;15(8):539–546. doi: 10.1002/bies.950150807. [DOI] [PubMed] [Google Scholar]
  39. Landsman D. No HMG-1 box signature. Nature. 1993 Jun 17;363(6430):590–590. doi: 10.1038/363590b0. [DOI] [PubMed] [Google Scholar]
  40. Laudet V., Stehelin D., Clevers H. Ancestry and diversity of the HMG box superfamily. Nucleic Acids Res. 1993 May 25;21(10):2493–2501. doi: 10.1093/nar/21.10.2493. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Lilley D. M. DNA--protein interactions. HMG has DNA wrapped up. Nature. 1992 May 28;357(6376):282–283. doi: 10.1038/357282a0. [DOI] [PubMed] [Google Scholar]
  42. Martindale D. W. Codon usage in Tetrahymena and other ciliates. J Protozool. 1989 Jan-Feb;36(1):29–34. doi: 10.1111/j.1550-7408.1989.tb02679.x. [DOI] [PubMed] [Google Scholar]
  43. Ner S. S. HMGs everywhere. Curr Biol. 1992 Apr;2(4):208–210. doi: 10.1016/0960-9822(92)90541-h. [DOI] [PubMed] [Google Scholar]
  44. Pinna L. A. Casein kinase 2: an 'eminence grise' in cellular regulation? Biochim Biophys Acta. 1990 Sep 24;1054(3):267–284. doi: 10.1016/0167-4889(90)90098-x. [DOI] [PubMed] [Google Scholar]
  45. Rodriguez Alfageme C., Rudkin G. T., Cohen L. H. Isolation, properties and cellular distribution of D1, a chromosomal protein of Drosophila. Chromosoma. 1980;78(1):1–31. doi: 10.1007/BF00291907. [DOI] [PubMed] [Google Scholar]
  46. Roth S. Y., Allis C. D. Chromatin condensation: does histone H1 dephosphorylation play a role? Trends Biochem Sci. 1992 Mar;17(3):93–98. doi: 10.1016/0968-0004(92)90243-3. [DOI] [PubMed] [Google Scholar]
  47. Roth S. Y., Collini M. P., Draetta G., Beach D., Allis C. D. A cdc2-like kinase phosphorylates histone H1 in the amitotic macronucleus of Tetrahymena. EMBO J. 1991 Aug;10(8):2069–2075. doi: 10.1002/j.1460-2075.1991.tb07738.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Roth S. Y., Schulman I. G., Richman R., Cook R. G., Allis C. D. Characterization of phosphorylation sites in histone H1 in the amitotic macronucleus of Tetrahymena during different physiological states. J Cell Biol. 1988 Dec;107(6 Pt 2):2473–2482. doi: 10.1083/jcb.107.6.2473. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Saitou N., Nei M. The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol. 1987 Jul;4(4):406–425. doi: 10.1093/oxfordjournals.molbev.a040454. [DOI] [PubMed] [Google Scholar]
  50. Schulman I. G., Wang T., Wu M., Bowen J., Cook R. G., Gorovsky M. A., Allis C. D. Macronuclei and micronuclei in Tetrahymena thermophila contain high-mobility-group-like chromosomal proteins containing a highly conserved eleven-amino-acid putative DNA-binding sequence. Mol Cell Biol. 1991 Jan;11(1):166–174. doi: 10.1128/mcb.11.1.166. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Thomas G., Thorne B. A., Hruby D. E. Gene transfer techniques to study neuropeptide processing. Annu Rev Physiol. 1988;50:323–332. doi: 10.1146/annurev.ph.50.030188.001543. [DOI] [PubMed] [Google Scholar]
  52. White E. M., Allis C. D., Goldfarb D. S., Srivastva A., Weir J. W., Gorovsky M. A. Nucleus-specific and temporally restricted localization of proteins in Tetrahymena macronuclei and micronuclei. J Cell Biol. 1989 Nov;109(5):1983–1992. doi: 10.1083/jcb.109.5.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Wu M., Allis C. D., Gorovsky M. A. Cell-cycle regulation as a mechanism for targeting proteins to specific DNA sequences in Tetrahymena thermophila. Proc Natl Acad Sci U S A. 1988 Apr;85(7):2205–2209. doi: 10.1073/pnas.85.7.2205. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Wu M., Allis C. D., Richman R., Cook R. G., Gorovsky M. A. An intervening sequence in an unusual histone H1 gene of Tetrahymena thermophila. Proc Natl Acad Sci U S A. 1986 Nov;83(22):8674–8678. doi: 10.1073/pnas.83.22.8674. [DOI] [PMC free article] [PubMed] [Google Scholar]

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