The CCR4 transcriptional regulatory complex consisting of CCR4, CAF1, DBF2 and other unidentified factors is one of several groups of proteins that affect gene expression. Using mass spectrometry, we have identified the 195, 185 and 116 kDa species which are part of the CCR4 complex. The 195 and 185 kDa proteins were found to be NOT1 and the 116 kDa species was identical to NOT3. NOT1, 2, 3 and 4 proteins are part of a regulatory complex that negatively affects transcription. All four NOT proteins were found to co-immunoprecipitate with CCR4 and CAF1, and NOT1 co-purified with CCR4 and CAF1 through three chromatographic steps in a complex estimated to be 1.2x10(6) Da in size. Mutations in the NOT genes affected many of the same genes and processes that are affected by defects in the CCR4 complex components, including reduction in ADH2 derepression, defective cell wall integrity and increased sensitivity to monoand divalent ions. Similarly, ccr4, caf1 and dbf2 alleles negatively regulated FUS1-lacZ expression, as do defects in the NOT genes. These results indicate that the NOT proteins are physically and functionally part of the CCR4 complex which forms a unique and novel complex that affects transcription both positively and negatively.

A process for the effective extraction and fractionation of phlorotannins from Fucus vesiculosus with high antioxidant potentials was investigated. The antioxidant activity of F. vesiculosus extract/fractions was assessed by 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging, reducing power, and ferrous ion-chelating assays. Among the crude extract and different polarity fractions, the phlorotannin-enriched ethyl acetate fraction possessed the highest DPPH scavenging activity and reducing power. This fraction was further fractionated by Sephadex LH-20 column chromatography or ultrafiltration. The antioxidant properties were evaluated by both the above chemical antioxidant tests and a mononuclear cell-based bioassay. Sephadex subfractions LH-2 and LH-3 with high total phlorotannin content exhibited strong DPPH quenching activity, comparable to those of ascorbic acid and butylated hydroxytoluene and significantly higher than that of α-tocopherol. Polyphenols in F. vesiculosus were found to consist mainly of high molecular weight phlorotannin polymers. There were no clear relationships between the degree of polymerization, molecular size, and antioxidant activity. All the subfractions separated by Sephadex LH-20 column chromatography and ultrafiltration showed a high ability to scavenge reactive oxygen species generated by mononuclear cells. Further characterization of the phlorotannin compounds was performed on six Sephadex subfractions. Several phlorotannin oligomers were tentatively identified on the basis of HPLC-ESI-MS(n) analyses.

The carbon sink capacity of tropical forests is substantially affected by tree mortality. However, the main drivers of tropical tree death remain largely unknown. Here we present a pan-Amazonian assessment of how and why trees die, analysing over 120,000 trees representing > 3800 species from 189 long-term RAINFOR forest plots. While tree mortality rates vary greatly Amazon-wide, on average trees are as likely to die standing as they are broken or uprooted—modes of death with different ecological consequences. Species-level growth rate is the single most important predictor of tree death in Amazonia, with faster-growing species being at higher risk. Within species, however, the slowest-growing trees are at greatest risk while the effect of tree size varies across the basin. In the driest Amazonian region species-level bioclimatic distributional patterns also predict the risk of death, suggesting that these forests are experiencing climatic conditions beyond their adaptative limits. These results provide not only a holistic pan-Amazonian picture of tree death but large-scale evidence for the overarching importance of the growth–survival trade-off in driving tropical tree mortality.

actions in this complex (Draper et al., 1994(Draper et al., , 1995. The 3 Corresponding author CAF1/POP2 protein has been identified as a component of the CCR4 complex (Draper et al., 1995), and caf1 CCR4, a general transcriptional regulator affecting disruptions display very similar phenotypes and transcripthe expression of a number of genes in yeast, forms a tional defects to those of ccr4 (Sakai et al., 1992; Draper multi-subunit complex in vivo. Using the yeast two et al., 1995). hybrid screen, we have identified DBF2, a cell cycleWe report here that DBF2 is another component of the regulated protein kinase, as a CCR4-associated protein.CCR4 complex. DBF2 was identified as a temperature-DBF2 is required for cell cycle progression at the sensitive mutation that causes cell cycle arrest at the end telophase to G 1 cell cycle transition. DBF2 co-immunoof mitosis in which the cells have a fully extended spindle precipitated with CCR4 and CAF1/POP2, a CCR4-and divided chromatin, a characterisitic of telophase associated factor, and co-purified with the CCR4 (Johnston et al., 1990). Consistent with a mitotic role for complex. Moreover, a dbf2 disruption resulted in DBF2, the gene is expressed under cell cycle control in phenotypes and transcriptional defects similar to those M phase. DBF2 encodes a protein kinase and this activity observed in strains deficient for CCR4 or CAF1. ccr4 is also cell cycle regulated, with a peak in late mitosis (Toyn and caf1 mutations, on the other hand, were found to and Johnston, 1994). Despite there being temperatureaffect cell cycle progression in a manner similar to sensitive alleles of DBF2, deletions of the gene are viable that observed for dbf2 defects. These data indicate that (Toyn et al., 1997) due to the existence of a homolog, DBF2 is involved in the control of gene expression and DBF20 (Toyn et al., 1991). However, deletion of both suggest that the CCR4 complex regulates transcription DBF2 and DBF20 results in strains that are non-viable, during the late mitotic part of the cell cycle.indicating that these genes encode closely related protein Keywords: CCR4/cell cycle/DBF2/protein kinase/ kinases that are essential for the ending of mitosis. The transcription target protein substrates of the DBF2 kinase have not been identified, and, therefore, the molecular basis for its role in regulation of the cell cycle is not known. Our present work indicates a role for DBF2 in transcriptional regula-

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