Human DNA ligase and DNA polymerase as molecular targets for heavy metals and anticancer drugs

DNA ligase and DNA polymerase play important roles in DNA replication, repair, and recombination. Frequencies of spontaneous and chemical- and physical-induced mutations are correlated to the fidelity of DNA replication. This dissertation elucidates the mechanisms of the DNA ligation reaction by DNA ligases and demonstrates that human DNA ligase I and DNA polymerase $\alpha$ are the molecular targets for two metal ions, Zn$\sp{2+}$ and Cd$\sp{2+},$ and an anticancer drug, F-ara-ATP.^ Human DNA ligases were purified to homogeneity and their AMP binding domains were mapped. Although their AMP-binding domains are similar, there could be difference between the two ligases in their DNA binding domains.^ The formation of the AMP-DNA intermediate and the successive ligation reaction by human DNA ligases were analyzed. Both reactions showed their substrate specificity for ligases I and II, required Mg2+, and were inhibited by ATP.^ A protein inhibitor from HeLa cells and specific for human DNA ligase I but not ligase II and T4 ligase was discovered. It reversibly inhibited DNA ligation activity but not the AMP-binding activity due to the formation of a reversible ligase I-inhibitor complex.^ F-ara-ATP inhibited human DNA ligase I activity by competing with ATP for the AMP-binding site of DNA ligase I, forming a ligase I-F-ara-AMP complex, as well as when it was incorporated at 3$\sp\prime$-terminus of DNA nick by DNA polymerase $\alpha.$^ All steps of the DNA ligation reaction were inhibited by Zn$\sp{2+}$ and Cd$\sp{2+}$ in a concentration-dependent manner. Both ions did not show the ability to change the fidelity of DNA ligation reaction catalyzed by human DNA ligase I. However, Zn$\sp{2+}$ and Cd$\sp{2+}$ showed their contradictory effects on the fidelity of the reaction by human DNA polymerase $\alpha.$ Zn$\sp{2+}$ decreased the frequency of misinsertion but less affected that of mispair extension. On the contrary, Cd$\sp{2+}$ increased the frequencies of both misinsertion and mispair extension at very low concentration. Our data provided strong evidence in the molecular mechanisms for the mutagenicity of zinc and cadmium, and were comparable with the results previously reported. ^

Effects of salinity, temperature and cadmium stress on cadmium -binding protein in the grass shrimp, {\it Palaemonetes pugio}

The combined effects of salinity, temperature and cadmium stress on survival and adaptation through cadmium-binding protein (CdBP) accumulation were studied in the grass shrimp, Palaemonetes pugio. In 96-hour bioassays, shrimp were exposed to zero or one of three levels of cadmium, under one of six different salinity (15, 25, or 35$\perthous$) and temperature (20 or 30$\sp\circ$C) regimes. CdBP concentrations were quantified in survivors from the 24 exposure groups. Salinity and temperature did not affect survivorship unless the shrimp were also exposed to cadmium. Grass shrimp were most sensitive to cadmium at low salinity-high temperature, and least sensitive at high salinity-low temperature. The incidence of cadmium-associated black lesions in gill tissue was influenced by salinity and temperature stress. P. pugio produced a 10,000 dalton metallothionein-like CdBP when exposed to at least 0.1 mg Cd$\sp{2+}$/L for 96 hours. Accumulation of CdBP was increased with increases in the exposure cadmium level, increases in temperature and decreases in salinity, independently and in conjunction with one another. Maximum CdBP concentrations occurred in grass shrimp that survived the salinity-temperature-cadmium conditions creating maximum stress as measured by highest mortality, not necessarily in shrimp exposed to the highest cadmium levels. The potential utility of this method as a monitor of physiological stress in estuarine biota inhabiting metal-polluted environments is discussed. ^

Mechanisms of multidrug resistance: Differences between natural and acquired adriamycin -resistant human colon carcinoma cells

Mechanisms of multidrug resistance (MDR) were studied in two independent MDR sublines (AdR1.2 and SRA1.2) derived from the established human colon carcinoma cell line LoVo. AdR1.2 was developed by long-term continuous exposure of the cells to adriamycin (AdR) in stepwise increments of concentration, while SRA1.2 was selected by repetitive pulse treatments with AdR at a single concentration. In this dissertation, the hypothesis that the mechanism of drug resistance in SRA1.2 is different than that in AdR1.2 is tested. While SRA1.2 demonstrated similar biological characteristics when compared to the parental LoVo, AdR1.2 exhibited remarkable alterations in biological properties. The resistance phenotype of AdR1.2 was reversible when the cells were grown in the drug-free medium whereas SRA1.2 maintained its resistance for at least 10 months under similar conditions. Km and Vmax of carrier-mediated facilitated diffusion AdR transport were similar among the three lines. However, both resistant sublines exhibited an energy-dependent drug efflux. AdR1.2 appeared to possess an activated efflux pump, and a decreased nucleus-binding of AdR, whereas SRA1.2 showed merely a lower affinity in binding of AdR to the nuclei. Southern blot analysis showed no amplification of the MDR1 gene in either of the two resistant subclones. However, Western blot analysis using the C219 monoclonal antibody against P170 glycoprotein detected a Mr 150-kDa plasma protein (P150) in AdR1.2 but not in SRA1.2 or in the parental LoVo. In vitro phosphorylation studies revealed that P150 was a phosphoprotein; its phosphorylation was Mg$\sp{2+}$-dependent and could be enhanced by verapamil, an agent capable of increasing intracellular AdR accumulation in AdR1.2 cells. The phosphorylation studies also revealed elevated phosphorylation of a Mr 66-kDa plasma membrane protein that was detectable in the AdR1.2 revertant and in AdR1.2 when verapamil was present, suggesting that hyperphosphorylation of the Mr 66-kDa protein may be related to the reversal of MDR. SDS-PAGE of the plasma membrane protein demonstrated overproduction of a Mr 130-kDa, MDR-related protein in both the resistant sublines. The Mr 130-kDa, MDR-related protein in both the resistant sublines. The Mr 130-kDa protein was not immunoreactive with C219, but its absence in the AdR1.2 revertant and the parental LoVo suggests that it is an MDR-related plasma membrane protein. In conclusion, the results from this study support the author's hypothesis that the mechanisms responsible for "Acquired" and "Natural" MDR are not identical. ^

Calmodulin -regulated processes and intracellular calcium in the heat stress response of mammalian cells

Three approaches were used to examine the role of Ca$\sp{2+}$- and/or calmodulin (CaM)-regulated processes in the mammalian heat stress response. The focus of the first approach was on the major Ca$\sp{2+}$-binding protein, CaM, and involved the use of CaM antagonists that perturbed CaM-regulated processes during heat stress. The second approach involved the use of a cell line and its BPV-1 transformants that express increased basal levels of CaM, or parvalbumin--a Ca$\sp{2+}$-binding protein not normally found in these cells. The last approach used Ca$\sp{2+}$ chelators to buffer Ca$\sp{2+}$-transients.^ The principle conclusions resulting from these three experimental approaches are: (1) CaM antagonists cause a temperature-dependent potentiation of heat killing, but do not inhibit the triggering and development of thermotolerance suggesting some targets for heat killing are different from those that lead to thermotolerance; (2) Members of major HSP families (especially HSP70) can bind to CaM in a Ca$\sp{2+}$-dependent manner in vitro, and HSP have been associated with events leading to thermotolerance. But, because thermotolerance is not affected by CaM antagonists, and antagonists should interfere with HSP binding to CaM, the events leading to triggering or developing thermotolerance were not strongly dependent on HSP binding to CaM; (3) CaM antagonists can also bind to HSP70 (and possibly other HSP) suggesting an alternative mechanism for the action of these agents in heat killing may involve direct binding to other proteins, like HSP70, whose function is important for survival following heating and inhibiting their activity; and (4) The signal governing the rate of synthesis of another major HSP group, the HSP26 family, can be largely abrogated by elevated Ca$\sp{2+}$-binding proteins or Ca$\sp{2+}$ chelators without significantly reducing survival or thermotolerance suggesting if the HSP26 family is involved in either end point, it may function in (Ca$\sp{2+}$) $\sb{\rm i}$ homeostasis. ^

Neocortical hyperexcitability defect in a mutant mouse model of spike-wave epilepsy, {\it stargazer}

Single-locus mutations in mice can express epileptic phenotypes and provide critical insights into the naturally occurring defects that alter excitability and mediate synchronization in the central nervous system (CNS). One such recessive mutation (on chromosome (Chr) 15), stargazer(stg/stg) expresses frequent bilateral 6-7 cycles per second (c/sec) spike-wave seizures associated with behavioral arrest, and provides a valuable opportunity to examine the inherited lesion associated with spike-wave synchronization.^ The existence of distinct and heterogeneous defects mediating spike-wave discharge (SWD) generation has been demonstrated by the presence of multiple genetic loci expressing generalized spike-wave activity and the differential effects of pharmacological agents on SWDs in different spike-wave epilepsy models. Attempts at understanding the different basic mechanisms underlying spike-wave synchronization have focused on $\gamma$-aminobutyric acid (GABA) receptor-, low threshold T-type Ca$\sp{2+}$ channel-, and N-methyl-D-aspartate receptor (NMDA-R)-mediated transmission. It is believed that defects in these modes of transmission can mediate the conversion of normal oscillations in a trisynaptic circuit, which includes the neocortex, reticular nucleus and thalamus, into spike-wave activity. However, the underlying lesions involved in spike-wave synchronization have not been clearly identified.^ The purpose of this research project was to locate and characterize a distinct neuronal hyperexcitability defect favoring spike-wave synchronization in the stargazer brain. One experimental approach for anatomically locating areas of synchronization and hyperexcitability involved an attempt to map patterns of hypersynchronous activity with antibodies to activity-induced proteins.^ A second approach to characterizing the neuronal defect involved examining the neuronal responses in the mutant following application of pharmacological agents with well known sites of action.^ In order to test the hypothesis that an NMDA receptor mediated hyperexcitability defect exists in stargazer neocortex, extracellular field recordings were used to examine the effects of CPP and MK-801 on coronal neocortical brain slices of stargazer and wild type perfused with 0 Mg$\sp{2+}$ artificial cerebral spinal fluid (aCSF).^ To study how NMDA receptor antagonists might promote increased excitability in stargazer neocortex, two basic hypotheses were tested: (1) NMDA receptor antagonists directly activate deep layer principal pyramidal cells in the neocortex of stargazer, presumably by opening NMDA receptor channels altered by the stg mutation; and (2) NMDA receptor antagonists disinhibit the neocortical network by blocking recurrent excitatory synaptic inputs onto inhibitory interneurons in the deep layers of stargazer neocortex.^ In order to test whether CPP might disinhibit the 0 Mg$\sp{2+}$ bursting network in the mutant by acting on inhibitory interneurons, the inhibitory inputs were pharmacologically removed by application of GABA receptor antagonists to the cortical network, and the effects of CPP under 0 Mg$\sp{2+}$aCSF perfusion in layer V of stg/stg were then compared with those found in +/+ neocortex using in vitro extracellular field recordings. (Abstract shortened by UMI.) ^

Inactivation and self -association of CaM kinase: Effects of ATP, substrate binding domains, and isozymic forms

Calcium/calmodulin-dependent protein kinase II (CaM kinase) is a multifunctional Ser/Thr protein kinase, that is highly enriched in brain and is involved in regulating many aspects of neuronal function. We observed that forebrain CaM kinase from crude homogenates, cytosolic fractions and purified preparations inactivates and translocates into the particulate fraction following autophosphorylation. Using purified forebrain CaM kinase as well as recombinant $\alpha$ isozyme, we determined that the formation of particulate enzyme was due to enzyme self-association. The conditions of autophosphorylation determine whether enzyme self-association and/or inactivation will occur. Self-association of CaM kinase is sensitive to pH, ATP concentration, and enzyme autophosphorylation. This process is prevented by saturating concentrations of ATP. However, in limiting ATP, pH is the dominant factor, and enzyme self-association occurs at pH values $\rm{<}7.0.$ Site-specific mutants were produced by substituting Ala for Thr286, Thr253, or Thr305,306 to determine whether these sites of autophosphorylation affect enzyme inactivation and self-association. The only mutation that influenced these processes was Ala286, which removed the protective effect afforded by autophosphorylation in saturating ATP. Enzyme inactivation occurs in the presence and absence of self-association and appears predominantly sensitive to nucleotide concentration, because saturating concentrations of $\rm Mg\sp{2+}/ADP$ or $\rm Mg\sp{2+}/ATP$ prevent this process. These data implicate the ATP binding pocket in both inactivation and self-association. We also observed that select peptide substrates and peptide inhibitors modeled after the autoregulatory domain of CaM kinase prevented these processes. The $\alpha$ and $\beta$ isozymes of CaM kinase were characterized independently, and were observed to exhibit differences in both enzyme inactivation and self-association. The $\beta$ isozyme was less sensitive to inactivation, and was never observed to self-associate. Biophysical characterization, and transmission electron microscopy coupled with image analysis indicated both isozymes were multimeric, however, the $\alpha$ and $\beta$ isozymes appeared structurally different. We hypothesize that the $\alpha$ subunit of CaM kinase plays both a structural and enzymatic role, and the $\beta$ subunit plays an enzymatic role. The ramifications for the functional differences observed for inactivation and self-association are discussed based on potential structural differences and autoregulation of the $\alpha$ and $\beta$ isozymes in both calcium-induced physiological and pathological processes. ^

Analysis of VirB4, a membrane-associated ATPase subunit essential for T -complex transport in Agrobacterium tumefaciens

Agrobacterium tumefaciens is a plant pathogen with the unique ability to export oncogenic DNA-protein complexes (T-complexes) to susceptible plant cells and cause crown gall tumors. Delivery of the T-complexes across the bacterial membranes requires eleven VirB proteins and VirD4, which are postulated to form a transmembrane transporter. This thesis examines the subcellular localization and oligomeric structure of the 87-kDa VirB4 protein, which is one of three essential ATPases proposed to energize T-complex transport and/or assembly. Results of subcellular localization studies showed that VirB4 is tightly associated with the cytoplasmic membrane, suggesting that it is a membrane-spanning protein. The membrane topology of VirB4 was determined by using a nested deletion strategy to generate random fusions between virB4 and the periplasmically-active alkaline phosphatase, $\sp\prime phoA$. Analysis of PhoA and complementary $\beta$-galactosidase reporter fusions identified two putative periplasmically-exposed regions in VirB4. A periplasmic exposure of one of these regions was further confirmed by protease susceptibility assays using A. tumefaciens spheroplasts. To gain insight into the structure of the transporter, the topological configurations of other VirB proteins were also examined. Results from hydropathy analyses, subcellular localization, protease susceptibility, and PhoA reporter fusion studies support a model that all of the VirB proteins localize at one or both of the bacterial membranes. Immunoprecipitation and Co$\sp{2+}$ affinity chromatography studies demonstrated that native VirB4 (87-kDa) and a functional N-terminally tagged HIS-VirB4 derivative (89-kDa) interact and that the interaction is independent of other VirB proteins. A $\lambda$ cI repressor fusion assay supplied further evidence for VirB4 dimer formation. A VirB4 dimerization domain was localized to the N-terminal third of the protein, as judged by: (i) transdominance of an allele that codes for this region of VirB4; (ii) co-retention of a His-tagged N-terminal truncation derivative and native VirB4 on Co$\sp{2+}$ affinity columns; and (iii) dimer formation of the N-terminal third of VirB4 fused to the cI repressor protein. Taken together, these findings are consistent with a model that VirB4 is topologically configured as an integral cytoplasmic membrane protein with two periplasmic domains and that VirB4 assembles as homodimers via an N-terminal dimerization domain. Dimer formation is postulated to be essential for stabilization of VirB4 monomers during T-complex transporter assembly. ^

Mid-Pliocene palynomorph record of the North Atlantic

The mid-Pliocene was an episode of prolonged global warmth and strong North Atlantic thermohaline circulation, interrupted briefly at circa 3.30 Ma by a global cooling event corresponding to marine isotope stage (MIS) M2. Paleoceanographic changes in the eastern North Atlantic have been reconstructed between circa 3.35 and 3.24 Ma at Deep Sea Drilling Project Site 610 and Integrated Ocean Drilling Program Site 1308. Mg/Ca ratios and d18O from Globigerina bulloides are used to reconstruct the temperature and relative salinity of surface waters, and dinoflagellate cyst assemblages are used to assess variability in the North Atlantic Current (NAC). Our sea surface temperature data indicate warm waters at both sites before and after MIS M2 but a cooling of ~2-3°C during MIS M2. A dinoflagellate cyst assemblage overturn marked by a decline in Operculodinium centrocarpum reflects a southward shift or slowdown of the NAC between circa 3.330 and 3.283 Ma, reducing northward heat transport 23-35 ka before the global ice volume maximum of MIS M2. This will have established conditions that ultimately allowed the Greenland ice sheet to expand, leading to the global cooling event at MIS M2. Comparison with an ice-rafted debris record excludes fresh water input via icebergs in the northeast Atlantic as a cause of NAC decline. The mechanism causing the temporary disruption of the NAC may be related to a brief reopening of the Panamanian Gateway at about this time.

Relative abundance and ranges of selected diatoms from Pliocene-Pleistocene sections of ODP Leg 177, Atlantic sector of the Southern Ocean

During Ocean Drilling Program (ODP) Leg 177, seven sites were drilled aligned on a transect across the Antarctic Circumpolar Current in the Atlantic sector of the Southern Ocean. The primary scientific objective of Leg 177 was the study of the Cenozoic paleoceanographic and paleoclimatic history of the southern high latitudes and its relationship with the Antarctic cryosphere development. Of special emphasis was the recovery of Pliocene-Pleistocene sections, allowing paleoceanographic studies at millennial or higher time resolution, and the establishment of refined biostratigraphic zonations tied to the geomagnetic polarity record and stable isotope records. At most sites, multiple holes were drilled to ensure complete recovery of the section. A description of the recovered sections and the construction of a multihole splice for the establishment of a continuous composite is presented in the Leg 177 Initial Reports volume for each of the sites (Gersonde, Hodell, Blum, et al., 1999). Here we present the relative abundance pattern and the stratigraphic ranges of diatom taxa encountered from shore-based light microscope studies completed on the Pliocene-Pleistocene sequences from six of the drilled sites (Sites 1089-1094). No shore-based diatom studies have been conducted on the Pliocene-Pleistocene sediments obtained at Site 1088, located on the northern crest of the Agulhas Ridge, because of the scattered occurrence and poor preservation of diatoms in these sections (Shipboard Scientific Party, 1999b). The data included in our report present the baseline of a diatom biostratigraphic study of Zielinski and Gersonde (2002), which (1) includes a refinement of the southern high-latitude Pliocene-Pleistocene diatom zonation, in particular for the middle and late Pleistocene, and (2) presents a biostratigraphic framework for the establishment of age models of the recovered sediment sections. Zielinski and Gersonde (2002) correlated the diatom ranges with the geomagnetic polarity record established shipboard (Sites 1090 and 1092) (Shipboard Scientific Party, 1999c, 1999d) and on shore (Sites 1089, 1091, 1093, and 1094) by Channell and Stoner (2002). The Pliocene-Pleistocene diatom zonation proposed by Zielinski and Gersonde (2002) relies on a diatom zonation from Gersonde and Bárcena (1998) for the northern belt of the Southern Ocean. Because of latitudinal differentiation of sea-surface temperature, nutrients, and salinity between Antarctic and Subantarctic/subtropical water masses, the Pliocene-Pleistocene stratigraphic marker diatoms are not uniformly distributed in the Southern Ocean (Fenner, 1991; Gersonde and Bárcena, 1998). As a consequence, Zielinski and Gersonde (2002) propose two diatom zonations for application in the Antarctic Zone south of the Polar Front (Southern Zonation, Sites 1094 and 1093) and the area encompassing the Polar Front Zone (PFZ) and the Subantarctic Zone (Northern Zonation, Sites 1089-1092). This accounts especially for the Pleistocene zonation where Hemidiscus karstenii, whose first abundant occurrence datum and last occurrence datum defines the subzonation of the northern Thalassiosira lentiginosa Zone, occurs only sporadically in the cold-water realm south of the PFZ and thus is not applicable in sections from this area. However, newly established marker species assigned to the genus Rouxia (Rouxia leventerae and Rouxia constricta) are more related to cold-water environments and allow a refinement of the Pleistocene stratigraphic zonation for the southern cold areas. A study relying on quantitative counts of both Rouxia species confirms the utility of these stratigraphic markers for the identification of sequences attributed to marine isotope Stages 6 and 8 in the southern Southern Ocean (Zielinski et al., 2002).

Mid-Cretaceous planktonic foraminifera off central Morocco

Sites 545 and 547 collectively penetrated 629 m of mid-Cretaceous strata (upper Aptian to upper Cenomanian) off central Morocco during Leg 79 of the Deep Sea Drilling Project. Site 545, at the base of the steep Mazagan Escarpment, records a virtually complete succession of hemipelagic sediments of early late Aptian to middle Cenomanian age. Minor faunal recycling occurred throughout much of the upper Aptian to middle Albian part of the sequence (Cores 55 through 41), reflecting bottom currents along the Mazagan Escarpment. This may be related to the strong upwelling regime and high surface water productivity over Site 545 during the latest Aptian through middle Albian. The upwelling system ceased rather abruptly in this area in late middle Albian time. Recycling of older strata by bottom currents also ceased in the late middle Albian and resulted in a slower average accumulation rate in the upper Albian to middle Cenomanian section of Site 545 (Cores 40 through 28). However, intervals of pebbly claystone conglomerates in Cores 40 and 34 record sporadic instability in the slope adjacent to Site 545. Site 547, located only about 15 km seaward, is situated in a small sub-basin adjacent to the basement block drilled by Site 544. It contains an expanded upper Albian to upper Cenomanian sequence as a result of the numerous conglomeratic intervals throughout much of the section. In contrast to Site 545, the conglomerates were not derived from older strata cropping out on the Mazagan Escarpment; rather, they originated penecontemporaneously from a local unstable slope. A detailed biostratigraphic framework based on planktonic foraminifers is established for the mid-Cretaceous sections of Sites 545 and 547 and a new composite zonal scheme is proposed for the early late Aptian through early late Cenomanian interval. Fifty-five species are recognized and illustrated

Eocene-Oligocene planktonic foraminifera of ODP Hole 135-841B

A middle Eocene to lower Oligocene sedimentary sequence was drilled at Site 841 in the Tonga forearc region during Ocean Drilling Program Leg 135. A 56-m-thick sequence of volcanic sandstone, spanning from Cores 135-841B-4IR to -47R (549.1 to 605 mbsf), unconformably overlies rhyolitic volcanic basement. The middle Eocene planktonic foraminifer assemblages (P Zone?), which occur in association with larger benthic foraminifers, include spinose species of Acarinina, Morozovella, and Truncorotaloides, but their abundance is low. Late Eocene and early Oligocene faunas are abundant and show the highest diversity of the Paleogene sequence drilled at this site. They have been assigned to Zones P15-16 and P18, respectively. The Eocene/Oligocene boundary was not recognized because of a hiatus in which Zone P17 (37.2-36.6 Ma) was missing. Another hiatus is recorded in the interval between the middle and late Eocene, spanning at least 1.8 Ma. Paleogene assemblages of Site 841 contain equal numbers of warm- and cool-water species, an attribute of the warm middle-latitude Paleogene fauna of the Atlantic Ocean. In particular, common to high abundances of cool-water taxa, such as Globorotaloides, Catapsydrax, Tenuitella, and small globigerinids, may be related to the opening of a shallow seaway south of Tasmania permitting the influx of cool Indian Ocean waters into the South Pacific before the late Eocene (approximately 37 Ma).

Sporomorphs and quantitative analysis of the Eocene record from IODP Hole 318-U1356A

The early Eocene epoch was characterized by extreme global warmth, which in terrestrial settings was characterized by an expansion of near-tropical vegetation belts into the high latitudes. During the middle to late Eocene, global cooling caused the retreat of tropical vegetation to lower latitudes. In high-latitude settings, near-tropical vegetation was replaced by temperate floras. This floral change has recently been traced as far south as Antarctica, where along the Wilkes Land margin paratropical forests thrived during the early Eocene and temperate Nothofagus forests developed during the middle Eocene. Here we provide both qualitative and quantitative palynological data for this floral turnover based on a sporomorph record recovered at Integrated Ocean Drilling Program (IODP) Site U1356 off the Wilkes Land margin. Following the nearest living relative concept and based on a comparison with modern vegetation types, we examine the structure and diversity patterns of the Eocene vegetation along the Wilkes Land margin. Our results indicate that the early Eocene forests along the Wilkes Land margin were characterized by a diverse canopy composed of plants that today occur in tropical settings; their richness pattern was similar to that of present-day forests from New Caledonia. The middle Eocene forests were characterized by a canopy dominated by Nothofagus and exhibited richness patterns similar to modern Nothofagus forests from New Zealand.

Quaternary and Paleogene calcareous nannofossils of ODP Leg 115

The occurrences of ten datum events for the Quaternary and top Pliocene nannofossils are identified at nine Leg 115 sites. A quantitative investigation of Paleogene nannofossils in 470 samples selected from 11 holes at 9 sites yielded 197 taxa, including one new species and 10 unidentified taxa that are likely to be new species. Regional differences in the timing of some biostratigraphically important events are recognized, and a set of datum events useful for biostratigra- phy in the tropical Indian Ocean is presented. Biogeographical differences are minor for Paleogene cores from the tropical sites (Sites 707-716); however, the Quaternary and late early Oligocene floras observed at the two subtropical sites (Sites 705 and 706) differ significantly from the corresponding floras of the tropical sites. Bathymetrically controlled dissolution is recognized by the reduction of species diversity in the Paleogene flora. Selective dissolution of nannofossils is also evidenced by the percentage reduction of three holococcolith taxa, Lanternithus minutus, Zygrhablithus bijugatus, and Holococcolith type A as well as by the increase of Coccolithus pelagicusand Cribrocentrum reticulatumin the deeper sites.

Fauna associated with Bathymodiolus azoricus mussel beds within hydrothermal fields of the Mid-Atlantic Ridge

Structure of assemblages associated with mussel aggregations of Bathymodiolus azoricus was investigated. Mussel beds were found on hydrothermal vent fields on the Mid-Atlantic Ridge (Menez Gwen, Lucky Strike, and Rainbow) at depths 850-2400 m. The community structure of the mussel bed assemblages varied between studied areas. Large number of species was unique to mussel beds of the Menez Gwen field; the most observed taxa were not specialized hydrothermal species. All other nonunique species were found within the Lucky Strike region. The lowest mussel assemblage structure evenness was observed in the shallowest Menez Gwen area (850 m depth). We assume that two types of mussel assemblages (nematode-dominated and copepod-dominated) exist within the Lucky Strike field. The assemblages of B. azoricus differ significantly from assemblages of B. thermophilus inhabiting Pacific hydrothermal vents.

Calcareous nannofossil species richness across the Paleocene-Eocene Thermal Maximum

The Paleocene-Eocene Thermal Maximum (PETM, ~5 million years ago) was an interval of global warming and ocean acidification attributed to rapid release and oxidation of buried carbon. We show that the onset of the PETM coincided with a prominent increase in the origination and extinction of calcareous phytoplankton. Yet major perturbation of the surface-water saturation state across the PETM was not detrimental to the survival of most calcareous nannoplankton taxa and did not impart a calcification or ecological bias to the pattern of evolutionary turnover. Instead, the rate of environmental change appears to have driven turnover, preferentially affecting rare taxa living close to their viable limits.