Df31 is a novel nuclear protein involved in chromatin structure in Drosophila melanogaster.
We originally isolated the Df31 protein from Drosophila embryo extracts as a factor which could decondense Xenopus sperm, by removing the sperm specific proteins and interacting with histones to facilitate their loading onto DNA. We now believe that this protein has a more general function in cellular DNA metabolism. The Df31 gene encodes a very hydrophilic protein with a predicted molecular mass of 18.5 kDa. Immunostaining showed that Df31 was present in a wide range of cell types throughout differentiation and in both dividing and non-dividing cells. In all cases the protein is present in large amounts, comparable with the level of nucleosomes. Injection of antisense oligonucleotides to lower the level of Df31 in embryos caused severe disruption of the nuclear structure. Large irregular clumps of DNA were formed, and in most cases the amount of DNA associated with each clump was more than that found in a normal nucleus. Immunofluorescence, cell fractionation, and formaldehyde cross-linking show that Df31 is associated with chromatin and that a significant fraction of it binds very tightly. It also shows the same binding characteristics when loaded onto chromatin in vitro. Chromatin fractionation shows that Df31 is tightly associated with nucleosomes, preferentially with oligonucleosomes. Despite this no differences were observed in the properties of nucleosomes loaded in the in vitro system in the presence and absence of Df31. These results suggest that Df31 has a role in chromosomal structure, most likely acting as a structural protein at levels of folding higher than that of nucleosomes.
Crevel
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G
Dept Biochemistry and Immunology, St Georges Hospital Medical School, Cranmer Terrace, London SW17 ORE, UK.
Huikeshoven
H
H
Cotterill
S
S
D000595:Amino Acid Sequence; D000818:Animals; D002453:Cell Cycle; D002457:Cell Extracts; D002458:Cell Fractionation; D002467:Cell Nucleus / Q000378:metabolism; D002843:Chromatin / Q000378:metabolism* / Q000502:physiology; D002868:Chromosomal Proteins, Non-Histone / Q000235:genetics / Q000378:metabolism*; D004247:DNA / Q000378:metabolism; D029721:Drosophila Proteins; D004331:Drosophila melanogaster / Q000235:genetics / Q000378:metabolism; D017344:Genes, Insect; D019476:Insect Proteins / Q000235:genetics / Q000378:metabolism*; D008845:Microinjections / Q000379:methods; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D009707:Nucleosomes / Q000378:metabolism; D016376:Oligonucleotides, Antisense; D012965:Sodium Chloride
D002457:Cell Extracts; D002843:Chromatin; D002868:Chromosomal Proteins, Non-Histone; C427466:Df31 protein, Drosophila; D029721:Drosophila Proteins; D019476:Insect Proteins; D009687:Nuclear Proteins; D009707:Nucleosomes; D016376:Oligonucleotides, Antisense; D012965:Sodium Chloride; D004247:DNA