Replication of heterochromatin and structure of polytene chromosomes.

Heterochromatin is characteristically the last portion of the genome to be replicated. In polytene cells, heterochromatic sequences are underreplicated because S phase ends before replication of heterochromatin is completed. Truncated heterochromatic DNAs have been identified in polytene cells of Drosophila and may be the discontinuous molecules that form between fully replicated euchromatic and underreplicated heterochromatic regions of the chromosome. In this report, we characterize the temporal pattern of heterochromatic DNA truncation during development of polytene cells. Underreplication occurred during the first polytene S phase, yet DNA truncation, which was found within heterochromatic sequences of all four Drosophila chromosomes, did not occur until the second polytene S phase. DNA truncation was correlated with underreplication, since increasing the replication of satellite sequences with the cycE(1672) mutation caused decreased production of truncated DNAs. Finally, truncation of heterochromatic DNAs was neither quantitatively nor qualitatively affected by modifiers of position effect variegation including the Y chromosome, Su(var)205(2), parental origin, or temperature. We propose that heterochromatic satellite sequences present a barrier to DNA replication and that replication forks that transiently stall at such barriers in late S phase of diploid cells are left unresolved in the shortened S phase of polytene cells. DNA truncation then occurs in the second polytene S phase, when new replication forks extend to the position of forks left unresolved in the first polytene S phase.

Histone H2A.Z is widely but nonrandomly distributed in chromosomes of Drosophila melanogaster.

Variant histones that differ in amino acid sequence from S-phase histones are widespread in eukaryotes, yet the structural changes they cause to nucleosomes and how those changes affect relevant cellular processes have not been determined. H2A.F/Z is a highly conserved family of H2A variants. H2Av, the H2A.F/Z variant of Drosophila melanogaster, was localized in polytene chromosomes by indirect immunofluorescence and in diploid chromosomes by chromatin immunoprecipitation. H2Av was widely distributed in the genome and not limited to sites of active transcription. H2Av was present in thousands of euchromatic bands and the heterochromatic chromocenter of polytene chromosomes, and the H2Av antibody precipitated both transcribed and nontranscribed genes as well as noncoding euchromatic and heterochromatic sequences. The distribution of H2Av was not uniform. The complex banding pattern of H2Av in polytene chromosomes did not parallel the concentration of DNA, as did the pattern of immunofluorescence using H2A antibodies, and the density of H2Av measured by immunoprecipitation varied between different sequences. Of the sequences assayed, H2Av was least abundant on 1. 688 satellite sequences and most abundant on the hsp70 genes. Finally, transcription caused, to an equivalent extent, both H2Av and H2A to be less tightly associated with DNA.