The Immune Inhibitor A1 Protease of Bacillus anthracis

Bacillus anthracis, an organism ubiquitous in the soil and the causative agent of anthrax, utilizes multiple mechanisms to regulate secreted factors; one example is the activity of secreted proteases. One of the most abundant proteins in the culture supernates of B. anthracis is the Immune Inhibitor A1 (InhA1) protease. Here, I demonstrate that InhA1 modulates the abundance of approximately half of the proteins secreted into the culture supernates, including substrates that are known to contribute to the ability of the organism to cause virulence. For example, InhA1 cleaves the anthrax toxin proteins, PA, LF, and EF. InhA1 also targets a number of additional proteases, including Npr599, contributing to a complex proteolytic regulatory cascade with far-reaching affects on the secretome. Using an intra-tracheal mouse model of infection, I found that an inhA-null strain is attenuated in relation to the parent strain. The data indicate that reduced virulence of the inhA mutant strain may be the result of toxin protein deregulation, decreased association with macrophages, and/or the inability to degrade host antimicrobial peptides. Given the significant modulation of the secretome by InhA1, it is likely that expression of the protease is tightly regulated. To test this I examined inhA1 transcript and protein levels in the parent and various isogenic mutant strains and found that InhA1 expression is regulated by several mechanisms. First, the steady state levels of inhA1 transcript are controlled by the regulatory protein SinR, which inhibits inhA1 expression. Second, InhA1 abundance is inversely proportional to the SinR-regulated protease camelysin, indicating the post-transcriptional regulation of InhA1 by camelysin. Third, InhA1 activity is dependent on a conserved zinc binding motif, suggesting that zinc availability regulates InhA1 activity. The convergence of these regulatory mechanisms signifies the importance of tight regulation of InhA1 activity, activity that substantially affects how B. anthracis interacts with its environment.

14-3-3 proteins interact with the beta-thymosin repeat protein Csp24.

Conditioned stimulus pathway protein 24 (Csp24) is a beta-thymosin-like protein that is homologous to other members of the family of beta-thymosin repeat proteins that contain multiple actin binding domains. Actin co-precipitates with Csp24 and co-localizes with it in the cytosol of type-B photoreceptor cell bodies. Several signal transduction pathways have been shown to regulate the phosphorylation of Csp24 and contribute to cellular plasticity. Here, we report the identification of the adapter protein 14-3-3 in lysates of the Hermissenda circumesophageal nervous system and its interaction with Csp24. Immunoprecipitation experiments using an antibody that is broadly reactive with several isoforms of the 14-3-3 family of proteins showed that Csp24 co-precipitates with 14-3-3 protein, and nervous systems stimulated with 5-HT exhibited a significant increase in co-precipitated Csp24 probed with a phosphospecific antibody as compared with controls. These results indicate that post-translational modifications of Csp24 regulate its interaction with 14-3-3 protein, and suggest that this mechanism may contribute to the control of intrinsic enhanced excitability.

Dissecting the Interaction between p53 and TRIM24

Dissecting the Interaction of p53 and TRIM24 Aundrietta DeVan Duncan Supervisory Professor, Michelle Barton, Ph.D. p53, the “guardian of the genome”, plays an important role in multiple biological processes including cell cycle, angiogenesis, DNA repair and apoptosis. Because it is mutated in over 50% of cancers, p53 has been widely studied in established cancer cell lines. However, little is known about the function of p53 in a normal cell. We focused on characterizing p53 in normal cells and during differentiation. Our lab recently identified a novel binding partner of p53, Tripartite Motif 24 protein (TRIM24). TRIM24 is a member of the TRIM family of proteins, defined by their conserved RING, B-box, and coiled coil domains. Specifically, TRIM24 is a member of the TIF1 subfamily, which is characterized by PHD and Bromo domains in the C-terminus. Between the Coiled-coil and PHD domain is a linker region, 437 amino acids in length. This linker region houses important functions of TRIM24 including it’s site of interaction with nuclear receptors. TRIM24 is an E3-ubiquitin ligase, recently discovered to negatively regulate p53 by targeting it for degradation. Though it is known that Trim24 and p53 interact, it is not known if the interaction is direct and what effect this interaction has on the function of TRIM24 and p53. My study aims to elucidate the specific interaction domains of p53 and TRIM24. To determine the specific domains of p53 required for interaction with TRIM24, we performed co-immuoprecipitation (Co-IP) with recombinant full-length Flag-tagged TRIM24 protein and various deletion constructs of in vitro translated GST-p53, as well as the reverse. I found that TRIM24 binds both the carboxy terminus and DNA binding domain of p53. Furthermore, my results show that binding is altered when post-translational modifications of p53 are present, suggesting that the interaction between p53 and TRIM24 may be affected by these post-translational modifications. To determine the specific domains of TRIM24 required for p53 interaction, we performed GST pull-downs with in vitro translated, Flag-TRIM24 protein constructs and recombinant GST-p53 protein purified from E. coli. We found that the Linker region is sufficient for interaction of p53 and TRIM24. Taken together, these data indicate that the interaction between p53 and TRIM24 does occur in vitro and that interaction may be influenced by post-translational modifications of the proteins.

Mechanisms of protein regulation in rat skeletal muscle during resistance exercise and training

The cellular mechanisms through which adult rat skeletal muscle protein is regulated during resistance exercise and training was investigated. A model of non-voluntary resistance exercise was described which involves the electrically-stimulated contraction of the lower leg muscles of anesthetized rats against a weighted pulley-bar. Muscle protein synthesis rates were measured by in vivo constant infusion of $\sp3$H-leucine following a single bout of resistance exercise. Specific messenger RNA levels were determined by dot-blot hybridization analysis using $\sp{32}$P-labelled DNA probes after a single bout and multiple bouts of phasic training. The effects of phasic training on increasing skeletal muscle mass was assessed. Between 12 and 36 hours following a single resistance exercise bout (24-192 contractions), total mixed and myofibril protein synthesis rates were significantly increase (32%-65%) after concentric (gastrocnemius m.) and eccentric (tibialis anterior m.) contractions. Eccentric contractions had greater effects on myofibril synthesis with more prolonged increases in synthesis rates. Lower numbers of eccentric than concentric contractions were required to increase synthesis. Cellular RNA was increased after exercise but the relative levels of skeletal $\alpha$-actin and cytochrome c mRNAs were unchanged. Since increases in synthesis rates exceeded increases in RNA, post-transcriptional mechanisms may be primarily responsible for increased protein synthesis after a resistance exercise bout. After 10-22 weeks of phasic eccentric resistance training, muscle enlargement (16%-30%) was produced in the tibialis anterior m. after all training paradigms examined. In contrast, gastrocnemius m. enlargement after phasic concentric training occurred after moderate (24/bout) but not after high (192/bout) repetition training. The absence of muscle growth in the gastrocnemius m. after high repetition training despite increased synthesis rates after the initial bout and RNA and possibly mRNA accumulation during training suggests a role for post-translational mechanisms (protein degradation) in the control of muscle growth in the gastrocnemius m. It is concluded that muscle protein during resistance exercise and training is regulated at several cellular levels. The particular response may be influenced by the exercise intensity and duration, the training frequency and the type of contractile work (eccentric vs. concentric) performed. ^

Characterization of osteopontin charge forms produced by osteoblasts

Osteopontin (OPN) is a highly-phosphorylated extracellular matrix protein localized in bone, kidney, placenta, T-lymphocytes, macrophages, smooth muscle of the vascular system, milk, urine, and plasma. In ROS 17/2.8 osteoblast-like osteosarcoma cells, 1,25-dihydroxyvitamin D3 [1,25(OH)2D 3] regulates OPN at the transcriptional level resulting in increased steady state mRNA levels and increased production of OPN protein, maximal at 48 hours. Using ROS 17/2.8 cells as an osteoblast model, OPN was purified from culture medium after three hour treatments of either vehicle (ethanol) or 1,25(OH)2D3 via barium citrate precipitation followed by immunoaffinity chromatography. ^ Here, further evidence of regulation of OPN by 1,25(OH)2D 3 at the posttranslational level is presented. Prior to the up-regulation of OPN at the transcriptional level, 1,25(OH)2D3 induces a shift in OPN isoelectric point (pI) detected on two-dimensional gels from pI 4.6 to pI 5.1. Loading equal amounts of [32P]-labeled OPN recovered from ROS 17/2.8 cells exposed to 1,25(OH)2D3 or vehicle alone for three hours reveals that the shift from pI 4.6 to 5.1 is the result of reduced phosphorylation. Using structural analogs to 1,25(OH) 2D3, analog AT [25-(OH)-16-ene-23-yne-D3], which triggers Ca2+ influx through voltage sensitive Ca2+ channels but does not bind to the vitamin D receptor, mimicked the OPN pI shift while analog BT [1,25(OH)2-22-ene-24-cyclopropyl-D 3], which binds to the vitamin D receptor but does not allow Ca 2+ influx, did not. Inclusion of the Ca2+ channel blocker nifedipine also blocks the charge shift conversion of OPN. Further analysis of the signaling pathway initiated by 1,25(OH)2D3 reveals that inhibition of the cyclic 3′,5′ -adenosine monophosphate-dependent kinase, protein kinase A, or inhibition of the cyclic 3′,5′-guanine monophosphate-dependent kinase, protein kinase G, also prevents the charge shift conversion. ^ Isolation of OPN from rat femurs and tibiae provides evidence for the existence of these two OPN charge forms in vivo, evidenced by differential migration on isoelectric focusing gels and sodium dodecyl sulfate-polyacrylamide gels. Peptide sequencing of rat long bone fractions revealed the presence of a presumed dentin specific protein, dentin matrix protein-1 (DMP-1). Western blot analysis confirmed the existence of DMP-1 in these fractions. ^ Using the OPN charge forms in functional assays, it was determined that the charge forms have differential roles in both cell surface and mineralization functions. In cell attachment assays and Ca2+ influx assays using PC-3 prostate cancer cells, the pI 5.1 charge form of OPN was found to permit binding and increase intracellular Ca2+ concentrations of PC-3 cells. The increase in intracellular Ca2+ concentration was found to be integrin αvβ3-dependent. In mineralization assays, the pI 4.6 charge form of OPN promoted hydroxyapatite formation, while the pI 5.1 charge form had improved Ca2+ binding ability. ^ In conclusion, these findings suggest that 1,25(OH) 2D3 regulates OPN not only at the transcriptional level, but also plays a role in determination of the OPN phosphorylation state. The latter involves a short term (less than three hours) treatment and is associated with membrane-initiated Ca2+ influx. Functional assays utilizing the two OPN charge forms reveal the dependence of OPN post-translational state on its function. ^

Glycosylation of TRPM4 and TRPM5 channels: molecular determinants and functional aspects

The transient receptor potential channel, TRPM4, and its closest homolog, TRPM5, are non-selective cation channels that are activated by an increase in intracellular calcium. They are expressed in many cell types, including neurons and myocytes. Although the electrophysiological and pharmacological properties of these two channels have been previously studied, less is known about their regulation, in particular their post-translational modifications. We, and others, have reported that wild-type (WT) TRPM4 channels expressed in HEK293 cells, migrated on SDS-PAGE gel as doublets, similar to other ion channels and membrane proteins. In the present study, we provide evidence that TRPM4 and TRPM5 are each N-linked glycosylated at a unique residue, Asn(992) and Asn(932), respectively. N-linked glycosylated TRPM4 is also found in native cardiac cells. Biochemical experiments using HEK293 cells over-expressing WT TRPM4/5 or N992Q/N932Q mutants demonstrated that the abolishment of N-linked glycosylation did not alter the number of channels at the plasma membrane. In parallel, electrophysiological experiments demonstrated a decrease in the current density of both mutant channels, as compared to their respective controls, either due to the Asn to Gln mutations themselves or abolition of glycosylation. To discriminate between these possibilities, HEK293 cells expressing TRPM4 WT were treated with tunicamycin, an inhibitor of glycosylation. In contrast to N-glycosylation signal abolishment by mutagenesis, tunicamycin treatment led to an increase in the TRPM4-mediated current. Altogether, these results demonstrate that TRPM4 and TRPM5 are both N-linked glycosylated at a unique site and also suggest that TRPM4/5 glycosylation seems not to be involved in channel trafficking, but mainly in their functional regulation.

Regulation of androgen biosynthesis - A short review and preliminary results from the hyperandrogenic starvation NCI-H295R cell model

Regulation of androgen production is poorly understood. Adrenarche is the physiologic event in mid-childhood when the adrenal zona reticularis starts to produce androgens through specific expression of genes for enzymes and cofactors necessary for androgen synthesis. Similarly, expression and activities of same genes and products are deregulated in hyperandrogenic disorders such as the polycystic ovary syndrome (PCOS). Numerous studies revealed involvement of several signaling pathways stimulated through G-protein coupled receptors or growth factors transmitting their effects through cAMP- or non-cAMP-dependent signaling. Overall a complex network regulates androgen synthesis targeting involved genes and proteins at the transcriptional and post-translational levels. Newest players in the field are the DENND1A gene identified in PCOS patients and the MAPK14 which is the kinase phosphorylating CYP17 for enhanced lyase activity. Next generation sequencing studies of PCOS patients and transcriptome analysis of androgen producing tissues or cell models provide newer tools to identify modulators of androgen synthesis.

A 2-D guinea pig lung proteome map

Guinea pigs represent an important model for a number of infectious and non-infectious pulmonary diseases. The guinea pig genome has recently been sequenced to full coverage, opening up new research avenues using genomics, transcriptomics and proteomics techniques in this species. In order to further annotate the guinea pig genome and to facilitate future pulmonary proteomics in this species we constructed a 2-D guinea pig proteome map including 486 protein identifications and post translational modifications (PTMs). The map has been up-loaded to the UCD 2D-PAGE open access database (http://proteomics-portal.ucd.ie/). Transit peptides, N-terminal acetylations and other PTMs are available via Peptideatlas (ftp://PASS00619:NM455hi@ftp.peptideatlas.org/). This dataset is associated with a research article published in the Journal of Proteomics [1].

Isotopic effects of nitrate photochemistry in snow: a field study at Dome C, Antarctica

Stable isotope ratios of nitrate preserved in deep ice cores are expected to provide unique and valuable information regarding paleoatmospheric processes. However, due to the post-depositional loss of nitrate in snow, this information may be erased or significantly modified by physical or photochemical processes before preservation in ice. We investigated the role of solar UV photolysis in the post-depositional modification of nitrate mass and stable isotoperatios at Dome C, Antarctica, during the austral summer of 2011/2012. Two 30 cm snow pits were filled with homogenized drifted snow from the vicinity of the base. One of these pits was covered with a plexiglass plate that transmits solar UV radiation, while the other was covered with a different plexiglass plate having a low UV transmittance. Samples were then collected from each pit at a 2–5 cm depth resolution and a 10-day frequency. At the end of the season, acomparable nitrate mass loss was observed in both pits for the top-level samples (0–7 cm) attributed to mixing with the surrounding snow. After excluding samples impacted by the mixing process, we derived an average apparent nitrogen isotopic fractionation (15" app/of role in driving the isotopic fractionation of nitrate in snow.We have estimated a purely photolytic nitrogen isotopic fractionation (15"photo) of -55.8 12.0 ‰ from the difference in the derived apparent isotopic ractionations of the two experimental fields, as both pits were exposed to similar physical processes except exposure to solar UV. This value is in close agreement with the 15" photo value of -47.9 6.8 ‰ derived in a laboratory experiment simulated for Dome C conditions (Berhanu et al., 2014). We have also observed an insensitivity of 15" with depth in the snowpack under the given experimental setup. This is due to the uniform attenuation of incoming solar UV by snow, as 15" is strongly dependent on the spectral distribution of the incoming light flux. Together with earlier work, the results presented here represent a strong body of evidence that solar UV photolysis is the most relevant post-depositional process modifying the stable isotope ratios of snow nitrate at low-accumulation sites, where many deep ice cores are drilled. Nevertheless, modeling the loss of nitrate in snow is still required before a robust interpretation of ice core records can be provided.

Cultivation strategies to enhance productivity of Pichia pastoris: A review.

Pichia pastoris, a methylotrophic yeast, is an established system for the production of heterologous proteins, particularly biopharmaceuticals and industrial enzymes. To maximise and optimise the production of recombinant products, recent molecular research has focused on numerous issues including the design of expression vectors, optimisation of gene copy number, co-expression of secretory proteins such as chaperones, engineering of glycosylation and secretory pathways, etc. However, the physiological effects of different cultivation strategies are often difficult to separate from the molecular effects of the gene construct (e.g., cellular stress through over-expression or incorrect post-translational processing). Hence, overall system optimisation is difficult, even though it is urgently required in order to describe and understand the behaviour of new molecular constructs. This review focuses on particular aspects of recombinant protein production related to variations in biomass growth and their implications for strain design and screening, as well as on the concept of rational comparisons between cultivation systems for the development of specific production processes in bioreactors. The relationship between specific formation rates of secreted recombinant proteins, qp, and specific growth rates, μ, has been analysed in a conceptual attempt to compare different systems, particularly those based on AOX1/methanol and GAP/glucose, and this has now evolved into a pivotal concept for bioprocess engineering of P. pastoris.

RNA polymerase II large subunit degradation induced by ecteinascidin 743: Molecular characterization and subsequent rational investigation of antitumor mechanisms in cancers with clinical response

Ecteinascidin 743 (Et-743), which is a novel DNA minor groove alkylator with a unique spectrum of antitumor activity, is currently being evaluated in phase II/III clinical trials. Although the precise molecular mechanisms responsible for the observed antitumor activity are poorly understood, recent data suggests that post-translational modifications of RNA polymerase II Large Subunit (RNAPII LS) may play a central role in the cellular response to this promising anticancer agent. The stalling of an actively transcribing RNAPII LS at Et-743-DNA adducts is the initial cellular signal for transcription-coupled nucleotide excision repair (TC-NER). In this manner, Et-743 poisons TC-NER and produces DNA single strand breaks. Et-743 also inhibits the transcription and RNAPII LS-mediated expression of selected genes. Because the poisoning of TC-NER and transcription inhibition are critical components of the molecular response to Et-743 treatment, we have investigated if changes in RNAPII LS contribute to the disruption of these two cellular pathways. In addition, we have studied changes in RNAPII LS in two tumors for which clinical responses were reported in phase I/II clinical trials: renal cell carcinoma and Ewing's sarcoma. Our results demonstrate that Et-743 induces degradation of the RNAPII LS that is dependent on active transcription, a functional 26S proteasome, and requires functional TC-NER, but not global genome repair. Additionally, we have provided the first experimental data indicating that degradation of RNAPII LS might lead to the inhibition of activated gene transcription. A set of studies performed in isogenic renal carcinoma cells deficient in von Hippel-Lindau protein, which is a ubiquitin-E3-ligase for RNAPII LS, confirmed the central role of RNAPII LS degradation in the sensitivity to Et-743. Finally, we have shown that RNAPII LS is also degraded in Ewing's sarcoma tumors following Et-743 treatment and provide data to suggest that this event plays a role in decreased expression of the Ewing's sarcoma oncoprotein, EWS-Fli1. Altogether, these data implicate degradation of RNAPII LS as a critical event following Et-743 exposure and suggest that the clinical activity observed in renal carcinoma and Ewing's sarcoma may be mediated by disruption of molecular pathways requiring a fully functional RNAPII LS. ^

p16-induced apoptosis in Ewing's sarcoma

The tumor suppressor p16 is a negative regulator of the cell cycle, and acts by preventing the phosphorylation of RB, which in turn prevents the progression from G1 to S phase of the cell cycle. In addition to its role in the cell cycle, p16 may also be able to induce apoptosis in some tumors. Ewing's sarcoma, a pediatric cancer of the bone and soft tissue, was used to study the ability of p16 to induce apoptosis due to the fact that p16 is often deleted in Ewing's sarcoma tumors and may play a role in the oncogenesis or progression of this disease. The purpose of these studies was to determine whether introduction of p16 into Ewing's sarcoma cells would induce apoptosis. We infected the Ewing's sarcoma cell line TC71, which does not express p16, with adenovirus- p16 (Ad-p16). Ad-p16 infection led to the production of functional p16 as measured by the induction of G1 arrest. Ad-p16 infection induced as much as a 100% increase in G1 arrest compared to untreated cells. As measured by propidium iodide (PI) and Annexin V staining, Ad-p16 was able to induce apoptosis to levels 20–30 fold higher than controls. Furthermore, Ad-p16 infection led to loss of RB protein before apoptosis could be detected. The loss of RB protein was due to post-translational degradation of RB, which was inhibited by the addition of the proteasome inhibitors PS-341 and NPI-0052. Downregulation of RB with si-RNA sensitized cells to Ad-p16-induced apoptosis, indicating that RB protects from apoptosis in this model. This study shows that p16 leads to the degradation of RB by the ubiquitin/proteasome pathway, and that this degradation may be important for the induction of apoptosis. Given that RB may protect from apoptosis in some tumors, apoptosis-inducing therapies may be enhanced in tumors which have lost RB expression, or in which RB is artificially inactivated. ^

Xp95 is phosphorylated within the proline rich domain during Xenopus oocyte maturation

Xp95 is the Xenopus ortholog of a conserved family of scaffold proteins that have in common an N-terminal Bro1 domain and a C-terminal proline rich domain (PRD). The regulation of this protein family is poorly understood. We previously showed that Xp95 undergoes a phosphorylation-dependant gel mobility shift during meiotic maturation of Xenopus oocytes, the only natural biological system in which post-translational modifications of this family has been demonstrated. Here we characterized Xp95 phosphorylation via two approaches. First, we tested a series of Xp95 fragments for the ability to gel-shift during oocyte maturation, and found that a fragment containing amino acids 705-786 is sufficient to cause a gel-shift. This fragment is within the N-terminal region of Xp95's PRD (N-PRD). Second, we purified phosphorylated Xp95 and by mass spectrometry found that a 5080 Da peptide which maps to N-PRD (amino acids 706-756) contains two phosphorylation sites, one of which is T745, within the conserved CIN85 binding motif. By in vitro protein interaction assays, we that T745 is critical for CIN85/Xp95 interaction, and that Xp95 phosphorylation correlates with loss of binding to CIN85. We also show that an Alix fragment (amino acids 604-789) also undergoes a gel-shift during oocyte maturation and during colcemid-induced mitotic arrest of HeLa cells. These findings indicate that Xp95/Alix is phosphorylated on the PRD during M phase induction and that the PRD phosphorylation regulates partner protein interaction. ^

Structure and biosynthesis of a pyruvate -dependent enzyme---phosphatidylserine decarboxylase from {\it Escherichia coli\/}

Phosphatidylserine decarboxylase of E. coli, a cytoplasmic membrane protein, catalyzes the formation of phosphatidylethanolamine, the principal phospholipid of the organism. The activity of the enzyme is dependent on a covalently bound pyruvate (Satre and Kennedy (1978) J. Biol. Chem. 253, 479-483). This study shows that the enzyme consists of two nonidentical subunits, $\alpha$ (Mr = 7,332) and $\beta$ (Mr = 28,579), with the pyruvate prosthetic group in amide linkage to the amino-terminus of the $\alpha$ subunit. Partial protein sequence and DNA sequence analysis reveal that the two subunits are derived from a proenzyme ($\pi$ subunit, Mr = 35,893) through a post-translational event. During the conversion of the proenzyme to the $\alpha$ and $\beta$ subunits, the peptide bond between Gly253-Ser254 is cleaved, and Ser254 is converted to the pyruvate prosthetic group at the amino-terminus of the $\alpha$ subunit (Li and Dowhan (1988) J. Biol. Chem. 263, 11516-11522).^ The proenzyme cannot be detected in cells carrying either single or multiple copies of the gene (psd), but can be observed in a T7 RNA polymerase/promoter and transcription-translation system. The cleavage of the wild-type proenzyme occurs rapidly with a half-time on the order of 2 min. Changing of the Ser254 to cysteine (S254C) or threonine (S254T) slows the cleavage rate dramatically and results in mutants with a half-time for processing of around 2-4 h. Change of the Ser254 to alanine (S254A) blocks the cleavage of the proenzyme. The reduced processing rate with the mutations of the proenzyme is consistent with less of the functional enzyme being made. Mutants S254C and S254T produce $\sim$15% and $\sim$1%, respectively, of the activity of the wild-type allele, but can still complement a temperature-sensitive mutant of the psd locus. Neither detectable activity nor complementation is observed by mutant S254A. These results are consistent with the hydroxyl-group of the Ser254 playing a critical role in the cleavage of the peptide bond Gly253-Ser254 of the pro-phosphatidylserine decarboxylase, and support the mechanism proposed by Snell and co-workers (Recsei and Snell (1984) Annu. Rev. Biochem. 53, 357-387) for the formation of the prosthetic group of pyruvate-dependent decarboxylases. ^

Preliminary results and documentation of sediment cores CRP2 and CRP-2A from the Ross Sea off Cape Roberts, Antarctica

The site for CRP-2, 14 km east of Cape Roberts (77.006°S; 163.719°E), was selected to overlap the early Miocene strata cored in nearby CRP-1, and to sample deeper into the east-dipping strata near the western margin ofe he Victoria Land Basin to investigate Palaeogene climatic and tectonic history. CRP-2 was cored from 5 to 57 mbsf (metres below the sea floor) (core recovery 91 %), with a deviation resulting in CRP-2A being cored at the same site. CRP-2A reached down to 624mbsf (recovery 95%), and to strata with an age of c. 33-35 Ma. Drilling took place from 16 October to 25 November 1998, on 2.0-2.2 m of sea ice and through 178 m of water. Core fractures and other physical properties, such as sonic velocity, density and magnetic susceptibility, were measured throughout the core. Down-hole logs for these and other properties were run from 63 to 167 mbsf and subsequently from 200 to 623 mbsf, although density and velocity data could be obtained only to 440 mbsf because of hole collapse. Sonic velocity averages c. 2.0 km S-1 for the upper part of the hole, but there is an sharp increase to c. 3.0 km s-1 and also a slight angular unconformity, at 306 mbsf, corresponding most likely to the early/late Oligocene boundary (c. 28-30 Ma). Velocity then increases irregularly to around 3.6 km s-1 at the bottom of the hole, which is estimated to lie 120 m above the V4/V5 boundary. The higher velocities below 306 mbsf probably reflect more extensive carbonate and common pyrite cementation, in patches, nodules, bedding-parallel masses and as vein infills. Dip of the strata also increases down-hole from 3° in the upper 300 in to over 10° at the bottom. Temperature gradient is 21° k-1. Over 2 000 fractures were logged through the hole. Borehole televiewer imagery was obtained for the interval from 200 to 440 mbsf to orient the fractures for stress field analysis. Lithostratigraphical descriptions on a scale of 1:20 are presented for the full length of the core, along with core box images, as a 200 page supplement to this issue. The hole initially passed through a layer of muddy gravel to 5.5 mbsf (Lithological Sub-Unit or LSU 1.1), and then into a Quaternary diatom-bearing clast-rich diamicton to 21 mbsf (LSU 2. l), with an interval of alternating compact diamicton and loose sand, and containing a rich Pliocene foraminiferal fauna, to 27 mbsf (LSU 2.2). The unit beneath this (LSU 3.1) has similar physical properties (sonic velocity, porosity, magnetic susceptibility) and includes diamictites of similar character to those of LSU 2.1 and 2.2, but an early Miocene (c. 19 Ma) diatom assemblage at 28 mbsf (top of LSU 3.1) shows that this sub-unit is part of the older section. The strata beneath 27 mbsf, primary target for the project, extend from early Miocene to perhaps latest Eocene age, and are largely cyclic glacimarine nearshore to offshore sediments. They are described as 41 lithological sub-units and interpreted in terms of 12 recurrent lithofacies. These are 1) mudstone, 2) inter-stratified mudstone and sandstone, 3) muddy very fine to coarse sandstone, 4) well-sorted stratified fine sandstone, 5) moderately to well-sorted, medium-grained sandstone, 6) stratified diamictite, 7) massive diamictite, 8) rhythmically inter-stratified sandstone and mudstone, 9) clast-supported conglomerate, 10) matrix-supported conglomerate, 11) mudstone breccia and 12) volcaniclastic sediment. Sequence stratigraphical analysis has identified 22 unconformity-bounded depositional sequences in pre- Pliocene strata. They typically comprise a four-part architecture involving, in ascending order, 1) a sharp-based coarse-grained unit (Facies 6,7,9 or 10), 2) a fining-upward succession of sandstones (Facies 3 and 4), 3) a mudstone interval (Facies l), in some cases coarsening upward to muddy sandstones (Facies 3), and 4) a sharp-based sandstone dominated succession (mainly Facies 4). The cyclicity recorded by the strata is interpreted in terms of a glacier ice margin retreating and advancing from land to the west, and of rises and falls in sea level. Analysis of sequence periodicity awaits afirmer chronology. However, apreliminary spectral analysis of magnetic susceptibility for a deepwater mudstone within one of the sequences (from 339 to 347 mbsf) reveals ratios between hierarchical levels that are similar to those of the three Milankovitch orbital forcing periodicities. The strata contain a wide range of fossils, the most abundant being marine diatoms. These commonly form up to 5% of the sediment, though in places the core is barren (notably between 300 and 412 mbsf). Fifty samples out of 250 reviewed were studied in detail. The assemblages define ten biostratigraphical zones, some of them based on local or as yet undescribed forms. The assemblages are neritic, and largely planktonic, suggesting that the sea floor was mostly below the photic zone throughout deposition of the corcd sequence. Calcareous nannofossils, representing incursions of ocean surface waters, are much less common (72 out of 183 samples examined) and restricted to mudstone intervals a few tens of metres thick, but are important for dating. Foraminifera are also sparse (73 out of 135 samples) and represented only by calcareous benthic species. Changing assemblages indicate a shift from inshore environments in the early Oligocenc to outer shelf in the late Oligocenc, returning to inshore in the early Miocene. Marine palynomorplis yielded large numbers of well-preserved forms from most of the 116 samples examined. The new in situ assemblagc found last year in CRP-1 is extended down into the late Oligocene and a further new assemblage is found in the early Oligoccnc. Many taxa are new, and cannot us yet contribute to an improved understanding of chronology or ecology. Marine invertebrate macrofossils, mostly molluscs and serpulid tubes, are scattered throughout the core. Preservation is good in mudstones but poor in other lithologies. Climate on land is reflected in the content of terrestrial palynomorphs, which are extremely scarce down to c. 300 mbsf. Some forms are reworked, and others represent a low growing sparse tundra with at least one species of Nothofagus. Beneath this level, a significantly greater diversity and abundance suggests a milder climate and a low diversity woody vegetation in the early Oligocene, but still far short of the richness found in known Eocene strata of the region. Sedimentary facies in the oldest strata also suggest a milder climate in the oldest strata cored, with indications of substantial glacial melt-water discharges, but are typical of a coldcr climate in late Oligocene and early Miocene times. Clast analyses from diamictites reveal weak to random fabrics, suggesting either lack of ice-contact deposition or post-depositional modification, but periods when ice grounded at the drill site are inferred from thin zones of in-situ brecciated rock and soft-sediment folding. These are more common above c. 300 mbsf, perhaps reflecting more extensive glacial advances during deposition of those strata. Erosion of the adjacent Transantarctic Mountains through Jurassic basalt and dolerite-intruded Beacon strata into basement rocks beneath is recorded by petrographical studies of clast and sand grain assemblages. Core below 310 mbsf contains a dominance of fine-grained Jurassic dolerite and basalt fragments along with Beacon-derived coal debris and rounded quartz grains, whereas the strata above this level have a much higher proportion of basement derived granitoids, implying that the large areas of the adjacent mountains had been eroded to basement by the end of the early Oligocene. There is little indication of rift-related volcanism below 310 mbsf. Above this, however, basaltic and trachytic tephras are common, especially from 280 to 200 mbsf, from 150 to 46 mbsf, and in Pliocene LSU 2.2 from 21 to 27 mbsf. The largest volcanic eruptions generated layers of coarse (up to 1 cm) trachytic pumice lapilli between 97 and 114 mbsf. The thickest of these (1.2 m at 112 mbsf) may have produced an eruptive column extending tens of km into the stratosphere. A source within a few tens of km of the drill site is considered most likely. Present age estimates for the pre-Pliocene sequence are based mainly on biostratigraphy (using mainly marine diatoms and to a lesser extent calcareous nannofossils), with the age of the tephra from 112 to 114 mbsf (21.44k0.05 Ma from 84 crystals by Ar-Ar) as a key reference point. Although there are varied and well-preserved microfossil assemblages through most of the sequence (notably of diatoms and marine palynomorphs), they comprise largely taxa either known only locally or as yet undescribed. In addition, sequence stratigraphical analysis and features in the core itself indicate numerous disconformities. The present estimate from diatom assemblages is that the interval from 27 to 130 mbsf is early Miocene in age (c. 19 to 23.5 Ma), consistent with the Ar-Ar age from 112 to 114 mbsf. Diatom assemblages also indicate that the late Oligocene epoch extends from c. 130 to 307 mbsf, which is supported by late Oligocene nannofossils from 130 to 185 mbsf. Strata from 307 to 412 mbsf have no age-diagnostic assemblages, but below this early Oligocene diatoms and nannofossils have been recovered. A nannoflora at the bottom of the hole is consistent with an earliest Oligocene or latest Eocene age. Magnetostratigraphical studies based on about 1000 samples, 700 of which have so far undergone demagnetisation treatment, have provided a polarity stratigraphy of 12 pre-Pliocene magnetozones. Samples above 270 mbsf are of consistently high quality. Below this, magnetic behaviour is more variable. A preliminary age-depth plot using the Magnetic Polarity Time Scale (MPTS) and constrained by biostratigraphical data suggests that episodes of relatively rapid sedimentation took place at CRP-2 during Oligocene times (c. 100 m/My), but that more than half of the record was lost in a few major and many minor disconformities. Age estimates from Sr isotopes in shell debris and further tephra dating are expected to lead to a better comparison with the MPTS. CRP-2/2A has recorded a history of subsidence of the Victoria Land Basin margin that is similar to that found in CIROS-170 km to the south, reflecting stability in both basin and the adjacent mountains in late Cenozoic times, but with slow net accumulation in the middle Cenozoic. The climatic indicators from both drill holes show a similar correspondence, indicating polar conditions for the Quaternary but with sub-polar conditions in the early Miocene-late Oligocene and indications of warmer conditions still in the early Oligocene. Correlation between the CRP-2A core and seismic records shows that seismic units V3 and V4, both widespread in the Victoria Land Basin, represent a period of fluctuating ice margins and glacimarine sedimentation. The next drill hole, CRP-3, is expected to core deep into V5 and extend this record of climate and tectonics still further back in time.