Expression of a marked H2A histone protein in mammalian cells.
Histone protein sequences are highly conserved. In order to determine whether histones with sequences not found in nature would be tolerated in the chromatin of tissue culture cells, a gene for histone H2A.1a was altered by extending the protein coding region with eight amino acids, including three residues for methionine which are lacking in H2A.1a. Isolated clones of HeLa cells transfected with the gene construct were found to produce a novel protein which was resolved from other histone proteins on AUT-AUC two-dimensional gels. One clone, HeLa-B4, in which the novel protein named H2A.E accounted for about 10% of total H2A protein, was studied further. The linkage of H2A.E mRNA concentrations to the rates of DNA and protein synthesis was found to be the same as that of other replication-linked histone mRNA species. The stability of H2A.E in chromatin as well as the partitioning of nascent H2A.E protein between soluble and nuclear fractions was found to be indistinguishable from that of other histone species. This study shows that histone proteins with sequences other than the conserved sequences found in nature may be utilized in tissue culture cells.
Ivanova
V S
VS
Laboratory of Molecular Pharmacology, Developmental Therapeutics Program, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892.
Bonner
W M
WM
D000595:Amino Acid Sequence; D001483:Base Sequence; D002843:Chromatin / Q000378:metabolism; D002999:Clone Cells; D017124:Conserved Sequence; D015180:Electrophoresis, Gel, Two-Dimensional; D015870:Gene Expression; D006367:HeLa Cells; D006657:Histones / Q000096:biosynthesis* / Q000302:isolation & purification; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D010957:Plasmids; D012333:RNA, Messenger / Q000032:analysis / Q000096:biosynthesis; D015183:Restriction Mapping; D013997:Time Factors; D014162:Transfection
D002843:Chromatin; D006657:Histones; D012333:RNA, Messenger