SEND PLM061.MAY MSG%"PALMER_SCIENCE",ASAHQ PSDN-1086 APRIL SCIENCE SITREP R 030324Z MAY 93 FROM: W. DETRICH, SSL P A L M E R S T A T I O N A N T A R C T I C A TELEMAIL::PALMER.STA PHONE/FAX: 011-874-150-3157 SPAN::PALMER@ATSVAX.SPAN TELEX: 5841503157 PNHG INTERNET::PALMER@ATSVAX.RSMAS.MIAMI.EDU !TO ASA SHEPHERD !TO SPOLE NSFREP !TO MCM ASASAT, ASALABM, ASASUP, NSFMGR CC E-MAIL::ANTARCTIC.OPS, NSF.DPP.OCEANOPS, P.PENHALE, T.DELACA, R.HANSON, E.HOFMANN, R.BOOTH, O.HOLM.HANSEN, G.MITCHELL, M.VERNET, M.HUNTLEY, R.WHRITNER, D.KARL, W.FRASER, B.SIDELL, W.DETRICH, M.KENNICUTT, K.DUNTON, W.STOCKTON, L.QUETIN, R.ROSS, Sea.Space, W.TRIVELPIECE, R.SMITH.UCSB, GMCC.BOULDER(pass to B.Mendonca), F.AZAM, R.RADTKE, S.WEILER, SPOLE, DUKE, NATPALMER, ASAHQ, VLF@STAR.STANFORD.EDU, NSFMCM (includes all McMurdo addresses) KARENTZD@ALM.ADMIN.USFCA.EDU, CHAPPELL@UCRACC.SPAN, J.PROSPERO, TFOSTER@UCSCC.UCSC.EDU, DALLUGE@ATMOS.OGI.EDU, N.SWANBERG, ENELSON%MINES.BITNET@SDS.SPAN, R.BIDIGARE, SRCFNSF@CMV.DSIR.GOVT.NZ (includes all CHCH addresses). Responding: Please insert in message, all CAPS, with the ! in column 1: !TO PAL SCIENCE, LABMANAGER, ADMIN, MANAGER S-036 ADAPTATIONS TO COUNTER DIFFUSIONAL CONSTRAINTS IN MUSCLES OF CHANNICHTHYID ICEFISHES. Bruce D. Sidell, University of Maine. FIELD TEAM: Bruce D. Sidell, Chris C. Cheng-DeVries, Stephen F. Norton Note: for description of fishing activities from R/V Polar Duke, please see the Sitrep for project S-037; all fishing activities for our projects are carried out jointly. In general, results of animal capture activities have been successful although the yield of Channichthyid icefishes (both number and species diversity) has been somewhat lower this year than in previous seasons. During the month of April, significant scientific progress was made on several fronts by our project. Major accomplishments are described briefly below: 1. Fresh tissues (heart ventricle and auricle, oxidative and glycolytic skeletal muscles, gonadal tissues) were harvested and flash frozen in liquid nitrogen for subsequent analyses in our CONUS laboratory from: approximately 19 Chionodraco rastrospinosus, 14 Chaenocephalus aceratus, 5 Champsocephalus gunnari, 2 Pseudochaenichthys georgianus. Thus, samples were secured from 4 of the 11 extant icefish genera. These tissues will be used for western and northern blot analyses for determining the extent of expression of myoglobin-encoding mRNA and myoglobin protein (Mb) in these species. High molecular wt genomic DNA will be extracted from the gonadal tissues harvested in addition to that prepared from fresh material on site (see below). 2. High molecular wt genomic DNA was prepared successfully from both gonadal tissues of Chionodraco rastrospinosus and liver tissue of Pseudochaenichthys georgianus. Suitability of this DNA for ultimate production of a genomic library at our CONUS laboratory was evaluated by agarose gel electrophoresis. DNA thus prepared was ethanol-precip itated and is being transported back to our home laboratory in this form. C. rastrospinosus and P. georgianus are the two species of icefishes examined to date that express both myoglobin mRNA and protein among the icefish species. 3. Glycolytic (white) muscle samples were both fixed for electron microscopic analyses and freeze-clamped for biochemical analyses from 10 specimens of Chaenocephalus aceratus. Both analytical procedures will be carried out subsequently in our CONUS laboratory and are aimed at determining the anatomical density of sarcoplasmic reticulum and specific titer of the ryanodine receptor Ca++-channel protein in the tissue. We hypothesize that density and yield of these elements from icefish tissue may be higher than seen in homologous tissue from warmer-bodied animals as an adaptation to overcome slow rates of molecular diffusion intracellularly at cold cell temperature. Both the sarcoplasmic reticulum and the ryanodine receptor are involved directly in determining the rate of muscle relaxation. 4. Methodologies were refined successfully for the preparation of isolated cellular nuclei from ventricular tissues of icefish species. Isolated nuclear preparations further were prepared and flash frozen from heart tissues of both C. aceratus (does not express myoglobin mRNA or protein) and C. rastrospinosus (does express both myoglobin mRNA and protein). These preparations will be returned frozen to our CONUS laboratory for use in nuclear run-on assays to assess the level of expression of the Mb-encoding gene locus and in DNase sensitivity assays to ascertain whether the Mb-encoding structural gene is present in condensed or open areas of chromatin. The methodology for production of isolated nuclei will be used further during cruise 93-4 for preparation of more nuclei. 5. A method newly developed by our laboratory for the estimation of both diffusion coefficient for oxygen (DO2) and solubility constant for oxygen (aO2) simultaneously from a single experimental record was implemented successfully with icefish oxidative muscles. This method will permit us to test much more efficiently our hypothesis that the high lipid content of icefish oxidative muscle may enhance the rate of transcellular oxygen movement in this tissue and thus compensate partially for the lack of intracellular (Mb) and extracellular (hemoglobin) respiratory pigments. To date, 10 successful runs have been completed with tissues from C. aceratus. As a positive control, similar determinations are being performed (3 to date) with the Mb and hemoglobin-expressing species Gobionotothen gibberifrons which also is characterized as having a low intracellular lipid content. On 24 April, field team members B. Sidell and C. DeVries departed Palmer Station for transit north to Punta Arenas on R/V Polar Duke. S. Norton remained at Palmer Station. Additional field participants M. Vayda and D. Barry will join Norton for cruise 93-4. As always, the exemplary efforts of Palmer Station personnel and crew, personnel and master of R/V Polar Duke have been essential to successful completion of our work. S-037 ASSEMBLY AND STABILITY OF MICROTUBULES FROM ANTARCTIC FISH AT LOW TEMPERATURES. H. William Detrich, Northeastern University, Boston, Massachusetts. FIELD TEAM: William Detrich, Martin Billger, Laura Camardella, Anthony Frankfurter, Stephen King, and Sandra Parker. During April we continued to study the structural and functional properties of the microtubule proteins [tubulins and microtubule-associated proteins (MAPs)] of Antarctic fishes. To support our work, specimens of several Antarctic fishes were collected during two fishing trips (1300 11 April - 0900 13 April and 1300 20 April - 0900 23 April), which were performed in conjunction with S-036. Additional specimens were collected by the crew of R/V Polar Duke during southbound transit from Punta Arenas (9 April) and from King George Island (15 April). During these trips, trawling operations were conducted at fishing grounds located near Livingston, Low, and Brabant Islands. The Livingston site, first identified by Sidell (S-036) as a potential ground in 1991, yielded small numbers of two species (the nototheniid Dissostichus mawsoni, and the icefish Pseudochaenichthys georgianus) that are rarely encountered at the latter two locations. However, severe weather conditions (southwesterly gales) throughout the month precluded extensive fishing at Livingston and Low Islands; approximately 80% of the total catch was derived from the East Dallmann Bay MSSSI (West shore of Brabant Island). The aggregate S-036/S-037 catch totalled approximately 500 Antarctic rockcods (Gobionotothen gibberifrons, Notothenia coriiceps, D. mawsoni), 100 icefishes (Chaenocephalus aceratus, Champsocephalus gunnari, Chionodraco rastrospinosus, P. georgianus), 12 dragonfishes (Parachaenichthys charcoti), and 10 skates (e.g., Bathyraja maccaini). Fishes were transported alive to Palmer Station where they were maintained in seawater aquaria (0 to -1.5 deg C). At Palmer, we prepared tubulin, MAPs (e.g., dynein), and nucleic acids (RNA) from brain, gonadal tissues, and erythrocytes of appropriate species by methods that we developed during previous field seasons. After sacrifice and use by our project, many of these fishes were provided to S-036 for the preparation of additional samples. During the past month we made significant progress toward our goal of understanding the structural and functional adaptations of the tubulins and MAPs of Antarctic fishes. Our accomplishments include: 1. Isolation of carboxy-terminal (C-terminal) peptides from brain tubulin of the Antarctic rockcod N. coriiceps. Brain tubulin, containing both the alpha and beta subunits, was digested by the protease trypsin, and the products were resolved on a Pharmacia Mono-Q column by high-performance liquid chromatography (HPLC). The elution profile of the fish C- terminal peptides differed significantly from the profile for C-terminal peptides from bovine tubulin. This observation, and results from immunohistochemical analyses, suggest strongly that fish and bovine brain tubulins differ in posttranslational polyglutamylation, consistent with our hypothesis that structural changes in the C-termini are responsible in part for the unique functional properties of Antarctic fish tubulins. 2. Purification of erythrocyte tubulins from Antarctic fishes. We have successfully developed a method for purification of tubulin from marginal bands of red blood cells from the rockcods N. coriiceps and G. gibberifrons. During May we will evaluate the assembly properties and structural features of this tubulin. Preliminary results suggest that the erythrocyte tubulin differs functionally and structurally from the egg and brain tubulins of Antarctic fishes that we have characterized previously. Thus, the erythrocyte protein may constitute a third functionally specific, cold-adapted tubulin from Antarctic fishes. These results provide strong support for the multi-tubulin hypothesis (i.e., the multiple tubulins of higher eukaryotes serve different functions in different tissues). 3. Characterization of the cytoskeletal organization of fish skin cells. During April we also developed methods to culture cells from primary explants of skin from N. coriiceps, G. gibberifrons, and C. aceratus. These cultures have been grown at 1 deg C in a CO2-independent medium. Cell preparations were stained with monoclonal and polyclonal antibodies specific for mammalian beta tubulin and several mammalian MAPs, followed by fluorescein-conjugated secondary antibodies. Immunofluorescence microscopy of these preparations (using Palmer's Zeiss Standard 25 microscope) revealed characteristic filamentous microtubule arrays. These preparations will be returned to CONUS for more detailed analysis. The successful application of this technology to Antarctic fish cell cultures will allow us to conduct future investigations of microtubule organization, function, and dynamics at the cellular level. Some of these studies will be initiated in May. 4. Purification and characterization of flagellar dyneins from Antarctic fishes. Dyneins are important mechanochemical MAPs that employ the energy released by ATP hydrolysis to power motile processes in eukaryotes, including the movement of sperm and the transport of organelles within cells. We seek to understand the adaptation of these enzymes to the cold temperatures experienced by marine poikilotherms of the Antarctic. During April we refined techniques, first developed in 1991, for the purification of dynein from sperm flagella of N. coriiceps. We have isolated separate fractions enriched in outer-arm dyneins and in inner-arm dyneins, characterized their subunit compositions, and made preliminary measurements of their ATPase activities. These studies will be continued in May. 5. Isolation of nucleic acids from Antarctic fishes. To support our molecular studies of tubulin and MAP genes, we have isolated RNA from several tissues (brain, testis, ovary, gill, liver, blood cells, spleen, heart, head kidney, etc.) of five Antarctic fishes (N. coriiceps, G. gibberifrons, C. aceratus, C. rastrospinosus, and P. charcoti). These RNAs will be used in our laboratory for production of cDNA libraries and for analysis of the tissue specificity of tubulin and MAP gene expression. During May we will continue to prepare RNA samples, and we will also purify high-molecular-weight genomic DNA from testes of representative fish species (for production of genomic libraries). On 24 April, project member M. Billger departed Palmer for Punta Arenas on board R/V Polar Duke. W. Detrich, L. Camardella, A. Frankfurter, S. King, and S. Parker remain at Palmer to continue our studies during May. We thank the captain and crew R/V Polar Duke and the personnel of Antarctic Support Associates for their able and generous assistance throughout this period. We anticipate a successful conclusion to our field season as we head into May. S-091 SEISMIC OBSERVATORY. U.S. Geological Survey. No personnel on station. System is being monitored by station science technician. Data is successfully being collected and prepared for retrograde. S-106 STANFORD VLF. U. Inan, Stanford University, Stanford, California. FIELD TEAM: Bill Trabucco. B. Trabucco arrived on 10 April to upgrade the VLF system to an IBM compatible digital data acquisition system. Due to unforeseen problems with the archiving software, the system required reconfiguration to acquire VLF data on 1/4" Ampex recorder and consecutive Betamax VCR recorders. Daily data continues to be collected. The VLF antenna was refitted and aligned, new anchor guides were mounted at each end of individual antenna loops. B. Trabucco departed for CONUS on 24 April. System is being operated by ASA science technician Andy Archer. S-275 UM/DOE ATMOSPHERIC MONITORING PROGRAM AT PALMER STATION. T. Snowdon, University of Miami; C. Sanderson/N. Chui, EML/DOE, N.Y. No personnel on station. System being run by ASA science technician. System continues to operate on a normal weekly schedule of calibration, background, and sample counts, with one sample filter being exposed for the duration of the week. T-312 TERASCAN SATELLITE IMAGING SYSTEM. R. Whritner, Scripps Institute. No personnel on station. System is being operated by station science technician. Satellite passes have been successfully and continuously archiving. Capture of NOAA-12 satellite telemetry has diminished. System graphics capability is still non-functional. T-313 UV MONITORING EXPERIMENT. C. Booth, Biospherical Instruments. No personnel on station. System is being operated by station science technician. The system is consistently and successfully collecting UV data. Problems with the calibrations scans are being evaluated and steps are being taken to ensure the integrity of the collected data. DETRICH/NESS 03012342.607 PLM061.MAY Action?