Core histone N-termini play an essential role in mitotic chromosome condensation.

We have studied the role of core histone tails in the assembly of mitotic chromosomes using Xenopus egg extracts. Incubation of sperm nuclei in the extracts led to the formation of mitotic chromosomes, a process we found to be correlated with phosphorylation of the N-terminal tail of histone H3 at Ser10. When the extracts were supplemented with H1-depleted oligosomes, they were not able to assemble chromosomes. Selective elimination of oligosome histone tails by trypsin digestion resulted in a dramatic decrease in their ability to inhibit chromosome condensation. The chromosome assembly was also inhibited by each of the histone tails with differing efficiency. In addition, we found that nucleosomes were recruiting through the flexible histone tails some chromosome assembly factors, different from topoisomerase II and 13S condensin. These findings demonstrate that histone tails play an essential role in chromosome assembly. We also present evidence that the nucleosomes, through physical association, were able to deplete the extracts from the kinase phosphorylating histone H3 at Ser10, suggesting that this kinase could be important for chromosome condensation.

Control of the histone-acetyltransferase activity of Tip60 by the HIV-1 transactivator protein, Tat.

Tip60, a cellular histone-acetyltransferase, is known to interact with the HIV-1-encoded transactivator protein, Tat. In this work, we show that the interaction of Tat with Tip60 efficiently inhibits the Tip60 histone-acetyltransferase activity. Besides its histone-acetyltransferase activity, Tip60 can undergo an autoacetylation which is not affected by Tat interaction. Our data show that Tip60 does not significantly influence Tat-dependent transcriptional activation of the 5'-LTR of HIV, suggesting that its interaction with Tat affects some intrinsic cellular process. We were then able to identify a cellular gene, Mn-dependent superoxide dismutase (Mn-SOD), that has a Tip60-dependent transcriptional activity. Interestingly, the simultaneous expression of Tat and Tip60 abolishes the effect of Tip60 on the activity of the Mn-SOD promoter. We postulate that the HIV-1 transactivator, Tat, in targeting Tip60 hinders the expression of cellular genes (such as Mn-SOD) which normally interfere with the efficient replication and propagation of the virus.

A new candidate region for the positional cloning of the XLP gene.

X-linked lymphoproliferative disease (XLP) is an inherited immunodeficiency characterised by selective susceptibility to Epstein-Barr virus and frequent association with malignant lymphomas chiefly located in the ileocecal region, liver, kidney and CNS. Taking advantage of a large bacterial clone contig, we obtained a genomic sequence of 197620 bp encompassing a deletion (XLP-D) of 116 kb in an XLP family, whose breakpoints were identified. The study of potential exons from this region in 40 unrelated XLP patients did not reveal any mutation. To define the critical region for XLP and investigate the role of the XLP-D deletion, detailed haplotypes in a region of approximately 20 cM were reconstructed in a total of 87 individuals from 7 families with recurrence of XLP. Two recombination events in a North American family and a new microdeletion (XLP-G) in an Italian family indicate that the XLP gene maps in the interval between DXS1001 and DXS8057, approximately 800 kb centromeric to the previously reported familial microdeletion XLP-D.

High-resolution mapping of the X-linked lymphoproliferative syndrome region by FISH on combed DNA.

X-linked lymphoproliferative syndrome is an inherited immunodeficiency for which the responsible gene is currently unknown. Several megabase-sized deleted regions mapping to Xq25 have been identified in XLP patients, and more recently a 130-kb deletion has been reported (Lamartine et al., 1996; Lanyi et al., 1996). To establish a physical map of this deleted region and to identify the XLP gene, two cosmid contigs were established (Lamartine et al., 1996). However, the physical map of this region is still uncompleted and controversial and three points remain unsolved: (1) the centromeric-telomeric orientation of the whole region, (2) the relative orientation of the two contigs, and (3) the size of the gap between the two contigs. To provide a definitive answer to these questions, high-resolution mapping by fluorescence in situ hybridization on combed DNA and molecular approaches were combined to establish the physical map of the XLP region over 600 kb. Our results identified a gap of 150 kb between the two contigs, established the relative orientation of one contig to the other, and determine the centromeric-telomeric orientation of the whole region. Our results show that the order of the marker over this region is: cen.1D10T7-DF83-DXS982.tel.

Molecular cloning and mapping of a human cDNA (PA2G4) that encodes a protein highly homologous to the mouse cell cycle protein p38-2G4.

We have identified a novel human gene with strong homology to the mouse Pa2g4 cell cycle gene. This novel gene (called PA2G4) belongs to a gene family with members in several chromosome regions: 3q24-q25, 6q22, 9q21, 12q13, 18q12, 20p12 and Xq25. A composite cDNA of 1697 nucleotides was isolated. The sequence of this cDNA predicts a protein of 394 amino acids. The deduced amino acid sequence of this human protein shows very strong homology to the mouse protein p38-2G4. The cDNA analyzed probably corresponds to a functional copy found at 12q13.