Identification through overexpression and tagging of the variant type of the mouse H1e and H1c genes.
Multiple, nonallelic forms of histone H1 are found in most mammalian tissues. Attempts to determine a functional significance to this diversity is hindered by a paucity of primary amino acid sequence data. Although numerous H1 genes have been cloned, the type of variant they encode cannot be determined by sequence analysis alone. We used transformation and overexpression methods to determine that two cloned mouse H1 genes, MH143 and MH175, encode H1c and H1e, respectively. Since these genes have been completely sequenced, these results establish the amino acid sequence of these variants. Assignment was accomplished by mutagenically "tagging" the genes by incorporation of a codon for methionine, which is not found in the major somatic H1 variants. These genes were placed under the control of the mouse metallothionein I promoter and introduced into 3T3 cells. Products of these mutagenized genes were detected by [35S]methionine labeling and identified by high performance liquid chromatography and sodium dodecyl sulfate-acid polyacrylamide gel electrophoresis. We were also able to induce major alterations in the normal stoichiometry of chromatin-associated H1 variants through overproduction of H1c and H1e. This had little effect on the growth properties of these transformants.
Brown
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Department of Biochemistry, University of Mississippi Medical Center, Jackson 39216-4505.
Sittman
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D016475:3T3 Cells; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D015870:Gene Expression; D014644:Genetic Variation; D006657:Histones / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D004915:Leukemia, Erythroblastic, Acute; D008715:Methionine / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D010957:Plasmids; D016133:Polymerase Chain Reaction; D014407:Tumor Cells, Cultured
D006657:Histones; D009838:Oligodeoxyribonucleotides; D008715:Methionine