The loss of chromosomal integrity from DNA double-strand breaks introduced into mammalian cells by ionizing radiation results in the specific phosphorylation of histone H2AX on serine residue 139, yielding a specific modified form named γ-H2AX. An antibody prepared to the unique region of human γ-H2AX shows that H2AX homologues are phosphorylated not only in irradiated mammalian cells but also in irradiated cells from other species, including Xenopus laevis, Drosophila melanogaster, and Saccharomyces cerevisiae. The antibody reveals that γ-H2AX appears as discrete nuclear foci within 1 min after exposure of cells to ionizing radiation. The numbers of these foci are comparable to the numbers of induced DNA double-strand breaks. When DNA double-strand breaks are introduced into specific partial nuclear volumes of cells by means of a pulsed microbeam laser, γ-H2AX foci form at these sites. In mitotic cells from cultures exposed to nonlethal amounts of ionizing radiation, γ-H2AX foci form band-like structures on chromosome arms and on the end of broken arms. These results offer direct visual confirmation that γ-H2AX forms en masse at chromosomal sites of DNA double-strand breaks. The results further suggest the possible existence of units of higher order chromatin structure involved in monitoring DNA integrity.

Histone H2AX is a ubiquitous member of the H2A histone family that differs from the other H2A histones by the presence of an evolutionarily conserved C-terminal motif, -KKATQASQEY. The serine residue in this motif becomes rapidly phosphorylated in cells and animals when DNA double-stranded breaks are introduced into their chromatin by various physical and chemical means. In the present communication we show that this phosphorylated form of H2AX, referred to as ␥-H2AX, appears during apoptosis concurrently with the initial appearance of high molecular weight DNA fragments. ␥-H2AX forms before the appearance of internucleosomal DNA fragments and the externalization of phosphatidylserine to the outer membrane leaflet. ␥-H2AX formation is inhibited by N-benzyloxycarbonyl-Val-AlaAsp-fluoromethyl ketone and the inhibitor of caspaseactivated DNase, and it is induced when DNase I and restriction enzymes are introduced into cells, suggesting that any apoptotic endonuclease is sufficient to induce ␥-H2AX formation. These results indicate that ␥-H2AX formation is an early chromatin modification following initiation of DNA fragmentation during apoptosis.

attained, the development was stopped with 5% acetic acid and, after 10 min, the gel was finally washed in distilled water and air-dried onto the glass support. Air-drying allows the long and thin gel to be easily submitted to photographic and/or digital recording using a sequencing gel-sized scanner, such as the HP ScanJet 4C/T (Hewlett-Packard, Palo Alto, CA, USA). This avoids wethandling or the need of an apparatus for gel-drying between cellophane sheets. Once examined and/or recorded, the dried gel can be detached from the glass plate after overnight rinsing with warm 5 mol/L NaOH and the latter reused after extensive washing with detergent, ethanol and distilled water.In our laboratory, we have applied the full-length electrophoresis technique several times to the study of the pattern of proteins synthesized by MDA-MB231 tumor cells in response to microenvironmental stimuli (data not reported). Figure 1 shows that the protein gels submitted to full-length electrophoresis exhibit a sensitive staining of protein bands (from 30-60, depending on acrylamide concentration) and a low level of background, which make them suitable for documentation and for software-assisted qualitative and quantitative analysis of the electrophoretic pattern. The number of resolved proteins cannot be compared to that obtained by 2-D electrophoresis(seethe2-D proteinmap from MDA-MB231cellsavailableonlineathttp://www.anl.gov/BIO/PMG/ projects/index_hbreast.html ).Nevertheless, full-length 1-D electrophoresis is a faster method that ( i ) does not require special consumables and expensive apparatus (such as ampholytes and IPG gel systems), ( ii ) improves the resolution of the pattern of polypeptides submitted to monodimensional electrophoresis and ( iii ) expands the applications of the DNA sequencing apparatus for protein studies.