Conditional knockout mice reveal distinct functions for the global transcriptional coactivators CBP and p300 in T-cell development.

The global transcriptional coactivators CREB-binding protein (CBP) and the closely related p300 interact with over 312 proteins, making them among the most heavily connected hubs in the known mammalian protein-protein interactome. It is largely uncertain, however, if these interactions are important in specific cell lineages of adult animals, as homozygous null mutations in either CBP or p300 result in early embryonic lethality in mice. Here we describe a Cre/LoxP conditional p300 null allele (p300flox) that allows for the temporal and tissue-specific inactivation of p300. We used mice carrying p300flox and a CBP conditional knockout allele (CBPflox) in conjunction with an Lck-Cre transgene to delete CBP and p300 starting at the CD4- CD8- double-negative thymocyte stage of T-cell development. Loss of either p300 or CBP led to a decrease in CD4+ CD8+ double-positive thymocytes, but an increase in the percentage of CD8+ single-positive thymocytes seen in CBP mutant mice was not observed in p300 mutants. T cells completely lacking both CBP and p300 did not develop normally and were nonexistent or very rare in the periphery, however. T cells lacking CBP or p300 had reduced tumor necrosis factor alpha gene expression in response to phorbol ester and ionophore, while signal-responsive gene expression in CBP- or p300-deficient macrophages was largely intact. Thus, CBP and p300 each supply a surprising degree of redundant coactivation capacity in T cells and macrophages, although each gene has also unique properties in thymocyte development.

Isolation of Halobacterium salinarum retrieved directly from halite brine inclusions.

Halite crystals were selected from a 186 m subsurface core taken from the Badwater salt pan, Death Valley, California to ascertain if halophilic Archaea and their associated 16S rDNA can survive over several tens of thousands of years. Using a combined microscope microdrill/micropipette system, fluids from brine inclusions were aseptically extracted from primary, hopper texture, halite crystals from 8 and 85 metres below the surface (mbls). U-Th disequilibrium dating indicates that these halite layers were deposited at 9,600 and 97,000 years before present (ybp) respectively. Extracted inclusions were used for polymerase chain reaction (PCR) analysis with haloarchaea-specific 16S rDNA primers or placed into haloarchaea culture medium. Enrichment cultures were obtained from 97 kyr halite crystal inclusion fluid and haloarchaea-containing prepared crystals (positive controls), whereas inclusions from crystals of 9.6 kyr halite and the haloarchaea-free halite crystals (negative controls) resulted in no growth. Phylogenetic analysis (16S rDNA) of the 97 kyr isolate, designated BBH 001, revealed a homology of 100% with Halobacterium salinarum. DNA-DNA hybridization experiments confirmed that BBH 001 was closely related to H. salinarum (81-75% hybridization) and its ascription to this haloarchaea species. The described method of retrieving particle-containing brine from fluid inclusions offers a robust approach for assessing the antiquity of microorganisms associated with evaporites.