2001â2002 - California Sea Grant - UC San Diego

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of HSV8-lsc varies considerably depending on the growth conditions. Expre sion under the control of the rbcL promoter5 UTR resolved on SDSPAGE, and HSV8-lsc visualized by Western blo ting with anti Flag antibody. A shown in Fig. 6, a cumulation anti-flag antibody (Right). (B) Proteins fromE. coli and C. reinhardt i expre sing the Hsv8-lsc antibody were separated intosoluble and insoluble pe lets by centrifugation. Twenty micrograms ofprotein were either stained with C oma sie blue (Left) orbloted to nitroce lulose membrane and decorated with an Fig. 4. Expre sion of HSV8-lsc proteins in bacteria and chloroplast. (A)Twenty microgramsofcrude protein from E. coli,WT C.reinhardt i,andtransgenic lines 10-6-3 and 20-4-4 were separated bySDSPAGE and either stained with C oma sie blue (Left) orbloted to nitroce lulose membrane and decorated with an the light phase, regardle s of the ce l density. Alternatively, The atpA promoter5 UTR in strain 10-6-3 drives fairly constant levels in strain 20-4-4, shows a marked increase in the a cumulation of antibody a the end of the dark phase or immediately after entering anti-flag antibody (Right). (C) Soluble proteins from C. reinhardt i transgenic line 10-6-3 were eithertreated with (Bme) or without(no Bme) reducing agent before separation onSDSPAGE. Proteins were blo ted tonitroce lulose membrane and decorated with anti-flag antibody. of HSV8-lsc production at 10 6 ce ls per ml in a lightdark cycle, yet shows a tremendous increase in lsc a cumulation on entering the were resuspended in Tris-bu fered saline, and the ce ls lysed by sonication. Soluble proteins were separated from insoluble chloroplast-expre sed protein along with that of the bacterialexpre sed HSV8-lsc. HSV8-lsc transgenic bacteria and algae at 10 6 ce ls per ml than at 10 7 ce ls per ml. These data clearly show that a cumulation of HSV8-lsc in chloroplast of C. reinhardt i can light phase when ce ls are cultured at 10 7 ce ls per ml. When grown under continuous light, both strains exhibited higher a cumulation by SDSPAGE, and HSV8-lsc proteins visualized by Western blot analysis. As shown in Fig. 4B, 60% of the HSV8-lsc proteins by centrifugation. Equal amounts of protein from the soluble fractions and from the insoluble pe lets were separated be optimized, dependent on the light regime used to culture the ce ls, the phase in the cycle at which ce ls are harvested, and the promoterUTR used to driv expre sion. and nonreducing gels. Under nonreducing conditions any disulfide bonds formed betw en the two heavy chain moieties of the antibody produced in bacteria partitioned to the insoluble fraction, whereas the HSV8-lsc produced in chloroplast was found exclusively in the soluble fraction. To determine whether chloroplast-expre sed antibodies contained any pos translational modifications we first examined the antibodies by SDSPAGE and Western blot analysis on reducing conditions chloroplast-expre sed HSV8-lsc runs as a much larger protein of 140 kDa, the size expected of a dimer. Treatment with should remain intact a lowing the antibody to migrate as a larger species. As shown in Fig. 4C (a rowhead), under nonreducing A PLIED BIOLOGICAL SCIENCES the monomer at 68 kDa. To ascertain whether any other pos translational modifications might be present in the chloroplast-expre sed BMe, to reduce disulfide bonds, results in the migration of the chloroplast HSV8-lsc proteins a the predicted molecular ma s of Fig.5. Characterization of HSV8-lscbinding to HSV8viralprotein via ELISA. Affinity-purified HSV8-lsc from the transgenic C. reinhardt i strains (10-6-3 transgeni chloroplast was functional, w examined the ability of chloroplast-expre sed HSV8-lsc to bind HSV8 proteins. HSV8-lsc proteins, we characterized the bacterial- and chloroplast-expre sed proteins by ma spectrometry. The ma spectra of peptide fragments from both the E. coli-and chloroplast-expre sed protein have an almost identical pa tern, indicating that no additional modifications are made to the chloroplast protein (data not shown). Fina ly, to determine whether the HSV8-lsc a cumulating in the hsv8-lsc were incubated inmicrotiter platescoated withaconstant amountof viral protein.Protein concentrationsinthese affinity-purifiedextractswere 13 ngl, of which 10% was HSV8-lsc as judged by C oma sie staining. Equal volumes ofWT C. reinhardt i proteins were used as a negative control (concentration 1 gl). and 16-3)were scr ened in an ELISA a say against HSVproteinsprepared from virus-infectedce ls. A totalof1 0,80,70, 60,30, 20,10,or5l of Flag-purified was purified from transgenic chloroplast by using an anti-flag a finity resin. As shown in Fig. 5, the chloroplast produced antibody recognized HSV8 proteins in ELISA a says in a robust manner. regimes on the a cumulation of antibody in the two transgenic strains, 10-6-3 and 20-4-4. Cultures of each strain were maintained Modulation of HSV8-lsc A cumulation in Transgenic Algae. To determine whether we could modulate the expre sion of HSV8-lsc in C. reinhardt i chloroplast, we examined the e fect of di ferent growth Mayfield et al. PNAS January 21, 2 03 vol. 1 0 no. 2 41 at 10 6 or 10 7 ce ls per ml and grown either in a 1212 lightdark cycle (5, 0 lux) or under continuous light (5, 0 lux). Ce ls were harvested by centrifugation and 20 g of soluble protein was communicating science to the public… From March 1, 2000 to February 28, 2002, California Sea Grant Communications produced 33 publications that describe on-going research projects and present the results of Sea Grant research and outreach activities. These publications were widely distributed to both scientific and lay audiences at state and national levels. Another 97 publications (including journal reprints, dissertation abstracts, technical reports, and conference proceedings) resulting from Sea Grantsponsored research and activities were made available to user communities and advertised via newsletters, publication lists and the web site. Two outreach projects each produced a video; a CALIFORNIA SEA GRANT Summary third project produced an electronic publication for Introduction the Internet; and a fourth produced a workbook for secondary school science teachers. Communications produced 31 additional publications, including newsletters, news releases, award and publication announcements, and brochures that were distributed to target audiences throughout the state and nationally. The Plant Journal (2002) 30(6), 733±744 TECHNICAL ADVANCE Development of a GFP reporter gene for Chlamydomonas reinhardtii chloroplast Program Directory 2002–2003 The California Sea Grant Extension Program partnered with other agencies to produce a 592-page comprehensive guide, “California’s Living Marine Resources: A Status Report.” The West Coast Ballast Outreach Project published and distributed to the shipping industry 13,000 copies of an educational poster, 17,000 copies of a brochure and several newsletters, which have literally been distributed around the world. Requests for the poster have come from as far away as Australia, India and Singapore. Much of California Sea Grant’s funded research is disseminated to the scientific community through General Public 21% Industry 16% Government 29% Education/Library 34% 40 publication in peer-reviewed, scientific journals. The Communications staff supplements this by advertising journal reprints to all its audiences and mailing single copies free of charge upon request. In order to decrease print and mailing costs and gain even broader dissemination, increasingly Sea Grant is making its publications available on its web site and notifying constituents of their availability. Scott Franklin, Binh Ngo, Ekem Efuet and Stephen P. May®eld * Department of Cell Biology and The Skaggs Institute for Chemical Biology, The Scripps Research Institute, 10550 N.Torrey Pines Road, La Jolla, CA 92037, USA Received 18 January 2002; revised 5 March 2002; accepted 6 March 2002. * For correspondence (fax +1 858 784 9840; e-mail sefrankl@scripps.edu). Reporter genes have been successfully used in chloroplasts of higher plants, and high levels of recombinant protein expression have been reported. Reporter genes have also been used in the chloroplast of Chlamydomonas reinhardtii, but in most cases the amounts of protein produced appeared to be very low. We hypothesized that the inability to achieve high levels of recombinant protein expression in the C. reinhardtii chloroplast was due to the codon bias seen in the C. reinhardtii chloroplast genome. To test this hypothesis, we synthesized a gene encoding green ¯uorescent protein (GFP) de novo, optimizing its codon usage to re¯ect that of major C. reinhardtii chloroplast-encoded proteins. We monitored the accumulation of GFP in C. reinhardtii chloroplasts transformed with the codon-optimized GFP cassette (GFPct), under the control of the C. reinhardtii rbcL 5¢- and 3¢-UTRs. We compared this expression with the accumulation of GFP in C. reinhardtii transformed with a nonoptimized GFP cassette (GFPncb), also under the control of the rbcL 5¢- and 3¢-UTRs. We demonstrate that C. reinhardtii chloroplasts transformed with the GFPct cassette accumulate »80-fold more GFP than GFPncb-transformed strains. We further demonstrate that expression from the GFPct cassette, under control of the rbcL 5¢- and 3¢-UTRs, is suf®ciently robust to report differences in protein synthesis based on subtle changes in environmental conditions, showing the utility of the GFPct gene as a reporter of C. reinhardtii chloroplast gene expression. Keywords: Chlamydomonas, GFP, reporter gene, codon bias. Reporter genes have greatly enhanced our ability to monitor gene expression in a number of biological organisms. In chloroplasts of higher plants, b-glucuronidase (uidA, Staub and Maliga, 1993); neomycin phosphotransferase (nptII, Carrer et al., 1993); adenosyl-3-adenyltransferase (aadA, Svab and Maliga, 1993); and the green ¯uorescent protein of Aequorea aequorea (gfp, Reed et al., 2001; Sidorov et al., 1999) have been used as reporter genes (Heifetz, 2000). Each of these genes has attributes that makes it a useful reporter of chloroplast gene expression, such as ease of analysis, sensitivity, or the ability to examine expression in situ. Based on these studies, other heterologous proteins have been expressed in the chloroplasts of higher plants, such as Bacillus thuringiensis Cry toxins, conferring resistance to insect herbivory (Kota et al., 1999), or human somatotropin (Staub et al., 2000), a potential biopharmaceutical. Several reporter genes have been expressed in the chloroplast of the eukaryotic green alga Chlamydomonas reinhardtii, although with varying degrees of success. These include aadA (Goldschmidt-Clermont, 1991; Zerges and Rochaix, 1994); uidA (Ishikura et al., 1999; Sakamoto et al., 1993); Renilla luciferase (Ren Luc, Minko et al., 1999); and the amino glycoside phosphotransferase from Acinetobacter baumanii, aphA6 (Bateman and Purton, 2000). The amount of recombinant protein produced was reported for the uidA gene only (Ishikura et al., 1999), and from these reports, and based on Western blot analysis or ã 2002 Blackwell Science Ltd 733 Publication requests by audience.
education… In order to promote the future wise use of our coastal resources, California Sea Grant invests in the next generation by providing education, training and informal learning opportunities for students from elementary through graduate school. GRADUATE TRAINEESHIPS Research projects funded by California Sea Grant generally include a stipend for at least one graduate student trainee who assists the project leader. The research done by the students is often the basis for their thesis. Hundreds of graduate students have been supported by the California Sea Grant trainee program since its inception in 1968. FELLOWSHIPS Fellowships in government provide first-hand training in solving today’s ocean and coastal resource problems. California Sea Grant’s fellowship programs offer unique opportunities for graduate students interested in marine resource policy. Sea Grant fellows participate in policy development and implementation and acquire the sociopolitical skills needed by today’s resource managers. Fellows benefit the legislative committees and government agencies they serve by supplying scientific knowledge of ocean and coastal resources. KNAUSS FELLOWSHIP California Sea Grant’s Knauss Fellows have been placed with a variety of host offices, including the Ocean Studies Board of the National Research Council, the Office of Fisheries Affairs of the U.S. Department of State, the Photo: William Fenical, Scripps Institution of Oceanography Division of Ocean Sciences of the National Science Foundation, the U.S. Fish and Wildlife Service, the House Committee on Resources, and NOAA Office of Global Programs. In 2000, Nelia Forest from UC Berkeley and Lisa Wooninck from UC Santa Barbara were awarded Knauss Fellowships. Forest worked at the Wetlands Division of the Environmental Protection Agency. Wooninck served at the Office of Congressman Sam Farr. In 2001, California Sea Grant’s nominees, Alix Cotumaccio of the Monterey Institute of International Studies and Kimberly Puglise of Moss Landing Marine Laboratories were awarded fellowships. They worked at the Coastal Ocean Program Office and the Office of Congressman Bart Stupak (D-MI), respectively. STATE FELLOWSHIP California Sea Grant sponsors a State Fellowship Program, modeled after the Knauss Fellowship, which places graduate students interested in statewide marine policy issues with host offices in the California legislature or state resource management agencies. State Fellows have worked with the state Joint Committee on Fisheries and Aquaculture, the Subcommittee for River Protection and Restoration, the Pacific Fisheries Legislative Task Force, the Monterey Bay National Marine Sanctuary, the California Regional Water Quality Control Board, and the California Coastal Commission. 41
Page 1 and 2: V alifornia Sea Grant Annual Report
Page 3: table of contents… Introduction .
Page 7 and 8: Mapping Essential Fish Habitat Alon
Page 9 and 10: “Fish populations around the coun
Page 11 and 12: The Abalone Farm may have as many a
Page 13 and 14: ecause it is older individuals that
Page 15 and 16: A beluga sturgeon, traditionally th
Page 17 and 18: Sea Grant Responds to Invasive Seaw
Page 19 and 20: survey, the California legislature
Page 21 and 22: A technician radios scientists to t
Page 23 and 24: account the influence of the large-
Page 25 and 26: of the alpha subclass of proteobact
Page 27 and 28: Under the agency’s orphan drug pr
Page 29 and 30: After 28 Years of Service, Seafood
Page 31 and 32: and Fishery Products.” These rule
Page 33 and 34: Santa Cruz & Monterey Counties RICH
Page 35 and 36: The goal is to understand what kind
Page 37 and 38: fall chinook over a 23-year period
Page 39 and 40: To educate the public about the new
Page 41: Nontoxic bottom paint being applied
Page 45 and 46: Discovery Center (ODC) and the Boys
Page 47 and 48: funding and allocations… Californ
Page 49 and 50: esidency at the University of Roche
Page 51 and 52: R/F-176 Radiometric Age Validation
Page 53 and 54: R/OE-37 Mitigation of Coastal Bluff
Page 55 and 56: Photo credit for cover (top to bott

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2001â2002 - California Sea Grant - UC San Diego