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Unlabelled: Eukaryotic cells respond to DNA damage and S phase replication blocks by arresting cell-cycle progression through the DNA structure checkpoint pathways. In Schizosaccharomyces pombe, the Chk1 kinase is essential for mitotic arrest and is phosphorylated after DNA damage. During S phase, the Cds1 kinase is activated in response to DNA damage and DNA replication blocks. The response of both Chk1 and Cds1 requires the six 'checkpoint Rad' proteins (Rad1, Rad3, Rad9, Rad17, Rad26 and Hus1). We demonstrate that DNA damage-dependent phosphorylation of Chk1 is also cell-cycle specific, occurring primarily in late S phase and G2, but not during M/G1 or early S phase. We have also isolated and characterized a temperature-sensitive allele of rad3. Rad3 functions differently depending on which checkpoint pathway is activated. Following DNA damage, rad3 is required to initiate but not maintain the Chk1 response. When DNA replication is inhibited, rad3 is required for both initiation and maintenance of the Cds1 response. We have identified a strong genetic interaction between rad3 and cds1, and biochemical evidence shows a physical interaction is possible between Rad3 and Cds1, and between Rad3 and Chk1 in vitro. Together, our results highlight the cell-cycle specificity of the DNA structure-dependent checkpoint response and identify distinct roles for Rad3 in the different checkpoint responses., Keywords: ATM/ATR/cell-cycle checkpoints/Chk1/Rad3

Chromosome 3q alterations occur frequently in many types of tumours. In a genetic screen for loci present in rhabdomyosarcomas, we identified an isochromosome 3q [i(3q)], which inhibits muscle differentiation when transferred into myoblasts. The i(3q) inhibits MyoD function, resulting in a non-differentiating phenotype. Furthermore, the i(3q) induces a 'cut' phenotype, abnormal centrosome amplification, aneuploidy and loss of G1 arrest following gamma-irradiation. Testing candidate genes within this region reveals that forced expression of ataxia-telangiectasia and rad3-related (ATR) results in a phenocopy of the i(3q). Thus, genetic alteration of ATR leads to loss of differentiation as well as cell-cycle abnormalities.

The primary structure of lipopolysaccharide binding protein (LBP), a trace plasma protein that binds to the lipid A moiety of bacterial lipopolysaccharides (LPSs), was deduced by sequencing cloned complementary DNA. LBP shares sequence identity with another LPS binding protein found in granulocytes, bactericidal/permeability-increasing protein, and with cholesterol ester transport protein of the plasma. LBP may control the response to LPS under physiologic conditions by forming high-affinity complexes with LPS that bind to monocytes and macrophages, which then secrete tumor necrosis factor. The identification of this pathway for LPS-induced monocyte stimulation may aid in the development of treatments for diseases in which Gram-negative sepsis or endotoxemia are involved.

The nucleotide sequence of approximately 3 kb of Bacillus subtilis DNA distal to the trp operon was determined. Three open reading frames were found and these were shown to encode the hisH, tyrA and aroE genes. Integrative plasmids were constructed to interrupt transcription through this region. These data suggest that these three genes can be transcribed from both the trp promoter preceding the trp operon and from a promoter within the trpA structural gene.

A sequence encoding bovine granulocyte-macrophage colony-stimulating factor (GM-CSF) has been identified from a concanavalin A-stimulated bovine lymphocyte cDNA library. This sequence was isolated by hybridization with synthetic oligonucleotide probes based upon the human GM-CSF sequence. This bovine cDNA was engineered for expression and secretion of activity into the periplasmic space of E. coli. Periplasmic extracts contain a 14,500-dalton protein and stimulate colony formation of bovine bone marrow progenitor cells. The predicted protein is 70% homologous with human GM-CSF and 55% homologous with murine GM-CSF. Numerous structural features are conserved among these three proteins, such as location of cysteine residues, glycosylation sites, and overall change. The biological activity of bovine GM-CSF is species specific, since recombinant preparations do not cause proliferation of human or murine bone marrow cells. Similarly, murine GM-CSF does not exhibit activity on cells of bovine or human origin. However, human GM-CSF does stimulate colony formation of bovine bone marrow cells, although the specific activity appears reduced when compared to assays on human cells.