New developments in marine biotechnology

Tai Lieu Chat Luong


New Developments in
Marine Biotechnology


New Developments in
Marine Biotechnology
Edited by

Y. LeGal
National Museum of Natural History and
College of France
Concarneau, France

and

H. 0. Halvorson
University of Massachusetts
Boston, Massachusetts

With the editorial assistance of

Anne-Marie Lambert

Springer Science+ Business Media, LLC


Library of Congress Cataloging-in-Publication Data

New developments 1n mar1ne biotechnology ' ed1ted by Y. LeGal and
H.O. Halvorson.
p.
em.
"Proceedings of the 4th International Mar1ne B1otechnology
Conference. held September 22-29. 1997, 1n Sorrento. Paestum,
Oranto, and Pugnochtuso. Italy''--T.p. verso.
Includes bib11ographical references anc.

1ndex.

ISBN 978-1-4419-3300-3
ISBN 978-1-4757-5983-9 (eBook)
DOI 10.1007/978-1-4757-5983-9

1. Marin~ flshes--Molecular aspects--Congresses. 2. Mar1ne
biotechnology--Congresses. 3. Fishery resources--Management-Congresses.
I. LeGal, Yves.
II. Halvorson. Harlyn D.
III. Internat1onal Marine Biotechnology Conference 14th
1997
Sorrento, Italy, etc.)
OL620.N49 1998
98-24800
572.8'1177--dc21
CIP

Proceedings of the 4th International Marine Biotechnology Conference,
held September 22-29, 1997, in Sorrento, Paestum, Otranto, and Pugnochiuso, Italy
ISBN 978-1-4419-3300-3

© 1998 Springer Science+ Business Media New York
Originally published by Plenum Press, New York in 1998
Softcover reprint of the hardcover 1st edition 1998

10987654321
All rights reserved
No part of this book may be reproduced, stored in a retrieval system, or transmitted in any form
or by any means, electronic, mechanical, photocopying, microfilming, recording, or otherwise,
without written permission from the Publisher


PREFACE

Past efforts to colonize the environment and domesticate living species, coupled
with scientific research, have resulted in the possession (but not always the real control)
by humans of any available terrestrial space. However, oceans, which represent up to twothirds of the surface of the planet, had not been really approached until the middle of this
century. As oceanographic science develops, the picture of a rich, diverse, complex and
also, in many respects, specific marine life, is coming into view.
In a broad sense, marine biotechnologies can be understood as the various means or
techniques of managing marine living systems for the benefit of mankind. The first goal
we have is for marine life to provide biomass for food. However, today it is not certain
that a significant increase of total world fisheries' catches will be possible in the future.
There are several ways to address this. First, we need to generate better, more complete, or
different uses of the biomass actually fished. This is mainly a matter of upgrading fish and
fish wastes. Second, we need to artificially grow the living species. This falls within the
scope of cell cultivation and of aquaculture. Both approaches have to be appreciated simultaneously in terms of biology, ecology, and economy. In both approaches, profit
improvements are linked to the introduction of biotechnological methods and to the use of
biotechnological processes.
The main characteristics of fished biomasses is that they still exist and are readily

available. They can be considered a huge reservoir of molecules: polysaccharides, enzymes, fats, etc., exhibiting physical, chemical, or biological activities of interest for various purposes. The main problem (and it is not a minor one), in terms of techniques and
cost, is to isolate and purify these molecules. The second issue in biomass treatment is
mass cultivation of marine organisms. It is now clear that trying to reproduce biomass intensively and artificially cannot easily yield profits, unless we use a series of biotechnological tricks that will permit a drastic lowering of the costs. During the last l 0 years,
another important problem has emerged. This is the spreading of pathogenic organisms in
overcrowded sea farms. Within a short period of time, sea farms could be almost completly destroyed by marine viruses, microorganisms, or parasites about which we have little information.
Solutions to these problems represent real strategic tasks for the marine biotechnologists requiring basic research in developmental biology, genetics, gene enginering, endocrinology, pathology, and immunology of species as different as flatfish, salmon, shrimps,
abalone, among others.
Biodiversity is largely a reflection of the very specific aspects of marine life. An
early trend consisted of limiting the scope of marine biotechnologies to the production of
v


vi

Preface

biological models that facilitate the study of general mechanisms. These studies feed our
knowledge and understanding of life that is built on an unique pattern. In contrast, they
also favor the exploitation of structural, developmental, and biochemical specificities.
Marine biotechnologies reveal their genuine potential in offering the investigation and exploitation of molecules and mechanisms for which we do not know of any terrestrial counterparts. Marine biotechnology is by nature multidisciplinary and clearly incorporates new
technologies from molecular biology and chemical analysis to bioreactor technology.
Marine biotechnologies also deal with environmental management. The first step in
any kind of management involves a diagnosis of the condition of a systems. The past decade has been marked by considerable progress in using rapid and sensitive methods for
estimating biological responses to human-induced changes in the environment. Many of
these methods now use molecular probes, nucleic acids, immunoreagents, or enzymatic
biosensors that allow us to record efficiently a considerable number of data. A main problem is how to handle this huge quantity of information, to use it, and to forecast the evolutionary trends of an estuary, a bay, a sea, or an ocean.
Finally, one of the most promising goals for marine biotechnologies will be the possibility of using sophisticated biological tools for managing marine ecosystems. Controlling natural production of useful species will be less costly than trying to rear completely
demanding species. Understanding the tenuousness of the relationship between planktonic
species and their environment will perhaps give us an insight on climatic changes and on
the biological future of the planet.

The domains covered by marine biotechnologies are vast and range over various
overlapping disciplines, from the molecular approaches of developmental biology and biodiversity to the chemistry of natural substances. New fields are rapidly evolving and are
helping to successively emphasize specific areas of biological sciences.
With its biphasic unfolding, the format of the fourth edition of International Marine
Biotechnology Conference (IMBC'97) was original and successful, as it enabled the presentation of straightforward reports and constructive discussions.
With more than sixty selected papers organized in eight sections, this book covers
the present state of the art in marine biotechnologies.
HHand YLG


TRIBUTE TO NINO SALVATORE

The International Marine Biotechnology Conferences represent an assembly of interdisciplinary scientists and technologists with a common interest in Marine Science. Nino
Salvatore was one of these. He joined the IOC to plan for IMBC'94 in Tromsoe, Norway
and quickly demonstrated that he was one of those rare individuals in the scientific community who made an almost instantaneous impression on any person fortunate enough to be
acquainted with him. His high standards and enthusiasm were widely felt-from the revitalizing of the Stazione Zoologica in Naples, to science policy in the EU, to support for
biotechnology, developmental biology, and molecular biology. Prof. Salvatore was a strong
enthusiast for basic research and its application to solving problems of the day.
During the IMBC'94 meeting, the lack of an European organization to deal with international and European collaboration became evident. Characteristically, Nino Salvatore
saw the need to establish such an organization. He organized an ad hoc meeting and a
decision was made to go ahead. The European Society for Marine Biotechnology was
formed, and its first President, Dr. Jan Olafsen, is a member of IOC and was our host in
Tromsoe for 1MBC'94.
When the decision was made to hold IMBC'97 in Italy, Dr. Salvatore applied his energy and enthusiam to its organization, financial support, and his wish to do something
different. An international program committee, chaired by Dr. Frank Gannon, developed a
program based on peer review of submitted abstracts. The mobility of the meeting is an
expression of Nino Salvatore's desire to have as many people and scenarios involved as
possible because of the diverse subject areas that need to be covered in biotechnology. If
people cannot come to the conference, the conference will visit them. He also had in mind
to permit as many of his countrymen to participate as possible while at the same time

broadening the picture of the scope of this interdisciplinary subject area in Italy in the
minds of foreign conference participants.
Science has lost a visionary person with a remarkable character. Individuals do make
a difference and Prof. Salvatore. He will be missed. The IMBC'97 is dedicated to him. We
seek your help in making this meeting a success and thereby honoring Gaetano Salvatore.
Harlyn 0. Halvorson

vii


ACKNOWLEDGMENTS

High Patronage of the President of the Italian Republic
Under the aegis of the European Union
Under the auspices of
Presidenza del Consiglio dei Ministri
Ministero dell 'Universita e Della Ric ere a Scientifica e Tecnologica
Ministero dei Beni Culturali e Ambientali
Consiglio nazionale delle Ricerche
Regione Campania
Regione Puglia
Amministrazione Provinciale di Napoli
Amministrazione Provinciale di Salerno
Amministrazione Provinciale di Foggia
Comune di Sorrento
Comune di Capaccio/Paestum
Comune di Otranto
Universita degli Studi di Napoli Federico II
Seconda Universita di Napoli
Universita degli Studi di Leece

Unione degli Industriali della Provincia di Napoli
With the support of
American Society for Microbiology
Biotechnology Center of Excellence Corp, USA
Department of Energy, USA
Massachusetts Foundation for Excellence in Marine and Polymer Science
National Science Foundation, USA
National Institutes of Health, USA
Office of Ocean and Atmospheric Research
Policy Center for Marine Biosciences and Technology, USA
Society for Industrial Microbiology, USA
United States Department of Agriculture
ix


Acknowledgments

X

With the contribution of'
Camera di Commercio Industria Artigianato E Agricoltura, Leece
Camera di Commercio Industria Artigianato E Agricoltura, Foggia
Ente Provinciale per il Turismo, Leece


CONTENTS

I. Biotechnology: Biology or Technology? Keynote Lecture
Arthur Kornberg

Section 1: Molecular Biology and Transgenic Animals
2. The Paradox of Growth Acceleration in Fish . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Jose de la Fuente, Isabel Guillen, and Mario P. Estrada

7

3. Gene Transfer in Zebrafish Enhanced by Nuclear Localization Signals . . . . . . . .
Philippe Collas and Peter Alestrom

II

4. Gene Transfer in Red Sea Bream (Pagrosomus major) . . . . . . . . . . . . . . . . . . . . .
Peijun Zhang, Yongli Xu, Zongzhu Liu, Yuan Xiang, Shaojun Du, and
ChoyL. Hew

15

5. Production of Lines of Growth Enhanced Transgenic Tilapia
(Oreochromis niloticus) Expressing a Novel Piscine Growth
Hormone Gene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Azirur Rahman and Norman Maclean
6. Retention of a Foreign Gene Transferred as a Protamine-DNA Complex by
Electroporated Salmon Sperm . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
F. Y. T. Sin, J. G. I. Khoo, U.K. Mukherjee, and I. L. Sin

19

29

Section 2: Natural Products and Processes

7. A Novel Antioxidant Derived from Seaweed . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
W. C. Dunlap, K. Masaki, Y. Yamamoto, R. M. Larsen, and I. Karube
8. Unusual Marine Sterols May Protect Cellular Membranes against Action of
Some Marine Toxins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Tatiana N. Makarieva, Valentine A. Stonik, Ludmila P. Ponomarenko, and
Dmitry L. Aminin

33

37

xi


xii

Contents

9. Secondary Metabolites of Marine Organisms . . . . . . . . . . . . . . . . . . . . . . . . . . . .
K. Mukesh, Miryam Z. Sahni, Valadmir Belenky Wahrman, and
Gurdial M. Sharma

41

I 0. Biosynthetic Studies on the Salinamides, Depsipeptides from a Marine
Streptomyces . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
·
Bradley S. Moore

49

II. Dereplication and Profiling of Marine Bacteria by Fatty Acid Analysis of Crude
Extracts Using Fourier Transform Mass Spectrometry . . . . . . . . . . . . . . . .
David J. Bourne, Eliane Abou-Mansour, Russell T. Hill, and Peter T. Murphy

55

12. Biocompatible Alginates for Use in Biohybrid Organs . . . . . . . . . . . . . . . . . . . . .
Gerd KlOck, Patrik Grohn, Christan Hasse, and Ulrich Zimmermann
13. Production ofBioactive Compounds by Cell and Tissue Cultures of Marine
Seaweeds in Bioreactor System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Gregory L. Rorrer, William H. Gerwick, and Donald P. Cheney
14. The Mermaid's Purse, or What the Skate Can Tell Us about Keeping Eggs Safe
in One Basket . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Thomas J. Koob, David P. Knight, Marina Paolucci, Bradley Noren, and
Ian P. Callard

61

65

69

15. In Vitro Production of Marine-Derived Antitumor Compounds . . . . . . . . . . . . . .
Shirley A. Pomponi, Robin Willoughby, Amy E. Wright, Claudia Pecorella,
Susan H. Sennett, Jose Lopez, and Gail Samples

73

16. Structure and Function of Barnacle Cement Proteins

Kei Kamino and Yoshikazu Shizuri

77

Section 3: Aquaculture
17. The Development and Commercialization of Tetraploid Technology for Oysters
Standish K. Allen, Jr., and Ximing Guo
18. New Technology for the Acceleration of Reproductive Maturation in
Economically Important Crustaceans . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Milton Fingerman, Rachakonda Sarojini, and Rachakonda Nagabhushanam
19. Endocrine Factors Regulating Crustacean Reproductive Maturation
Lei Liu and Hans Laufer
20. Studies on the Sea Bass Dicentrarchus labrax L. Immune System for Its Control
in Aquaculture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
G. Scapigliati, L. Abelli, N. Romano, L. Mastrolia, and M. Mazzini
21. Development of DNA Vaccines for Aquaculture . . . . . . . . . . . . . . . . . . . . . . . . . .
Joel Heppell, Tong Wu, Niels Lorenzen, Anthony E. Ellis, Susan M. Efler,
Neil K. Armstrong, Joachim Schorr, and Heather L. Davis

81

85

89

93
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xiii

Contents

22. Genetic Manipulation and Strain Improvement in Commercially Valuable Red
Seaweeds . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
D. Cheney, B. Rudolph, L. Z. Wang, B. Metz, K. Watson, K. Roberts, and
I. Levine

I 01

Section 4: Developmental Biology
23. Expression of Thyroid Hormone Receptor-a in the Growth and Development of
the Sea Bream (Sparus aurata) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Lynda Llewellyn, Vimi P. Ramsurn, Trevor Wigham, Deborah M. Power, and
Glen E. Sweeney
24. Regulation of Dlx Homeobox Gene Expression during Development of the
Zebrafish Embryo: The Potential Importance of Their Genomic
Organization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Marc Ekker, Genny Giroux, Ted Zerucha, Alison Lewis,
Adriana A. Gambarotta, and Joshua R. Schultz
25. Meiotic Cell Cycle Control by Mos in Ascidian Oocytes . . . . . . . . . . . . . . . . . . .
Gian Luigi Russo, Keiichiro Kyozuka, Marcella Marino, Elisabetta Tosti,
Martin Wilding, Maria Laura de Simone, and Brian Dale
26. Activation of Ciona intestinalis at Fertilisation Is Controlled by Nicotinamide
Nucleotide Metabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
M. Wilding, G. L. Russo, M. Marino, L. Grumetto, M. L. De Simone,
E. Tosti, and B. Dale

105

109

115

121

27. Apoptosis as a Normal Mechanism of Growth Control and Target of Toxicant
Actions during Spermatogenesis: Insights Using the Shark Testis Model . .
Gloria V. Callard, Leon M. McClusky, and Marlies Betka

125

28. Medakafish Embryonic Stem Cells as a Model for Genetic Improvement of
Aquaculture Livestocks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Yunhan Hong, Songlin Chen, Christoph Winkler, and Manfred Schartl

129

29. The Tropical Abalone Haliotis asinina as a Model Species to Investigate the
Molecular and Cellular Mechanisms Controlling Growth in Abalone
Regina T. Counihan, Nigel P. Preston, and Bernard M. Degnan

135

Section 5: Biology of Cell Factories
30. North American Porphyra Cultivation: From Molecules to Markets
I. A. Levine and D. Cheney

141

31. Oxygen Transport by Hemocyanins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Kensal E. van Holde

145

32. The Ink Gland of Sepia officina/is as Biological Model for Investigations of
Melanogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Anna Palumbo, Ida Gesualdo, Anna Di Cosmo, and Luigi De Martino

147


xiv

Contents

33. Recombinant Factor C from Carcinoscorpius rotundicauda Binds Endotoxin . . .
A. W. M. Pui, S. D. Roopashree, B. Ho, J. L. Ding

151

34. Molecular and Immunological Characterization of Shellfish Allergens
Patrick S. C. Leung and Ka-Hou Chu

155

35. Cell Cultures from the Abalone Haliotis tuberculata: A New Tool for in Vitro
Study of Biomineralization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
D. Sud, S. Auzoux-Bordenave, M. Martin, and D. Doumenc

165

Section 6: Bioremediation, Extremophiles, and Host-Pathogen Interactions
36. The Architecture ofDegradative Complex Polysaccharide Enzyme Arrays in a
Marine Bacterium Has Implications for Bioremediation . . . . . . . . . . . . . . .
Ronald Weiner, Devi Chakravorty, and Lynne Whitehead

37. Manganese Oxidation by Spores of the Marine Bacillus sp. Strain SG-1:

Application for the Bioremediation of Metal Pollution . . . . . . . . . . . . . . . .
Bradley M. Tebo, Lorraine G. van Waasbergen, Chris A. Francis,
Liming M. He, Deeanne B. Edwards, and Karen Casciotti

38. The Effects ofBioremediation on the Oil Degradation in Oil Polluted

Environments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Kim Sang-Jin, Jae Hak Sohn, Doo Suep Sim, Kae Kyoung Kwon, and
TaeHyunKim

39. Heavy Metal Binding Properties of Wild Type and Transgenic Algae
(Chlamydomonas sp.) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Xiao-Hua Cai, Jagat Adhiya, Samuel Traina, and Richard Sayre
40. DNA Repair Enzymes in Hyperthermophilic Archaea
Jocelyne DiRuggiero and Frank T. Robb
41. Chaperonin in a Thermophilic Methanogen, Methanococcus
thermolithotrophicus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Masahiro Furutani, Toshii Iida, and Shigeyuki Yamano, and
TadashiMaruyama
42. Production and Application of Natural Stabilizing Compounds from
Halotolerant Bacteria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Erwin A. Galinski and Thomas Sauer
43. Molecular Detection of Magnetic Bacteria in Deep-Sea Sediments
Kaori Inoue and Harald Petermann
44. Structure and Reaction Mechanism of the 13-Glycosidase from the Archaeon
Sulfolobus solfataricus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Marco Moracci, Maria Ciaramella, Laurence H. Pearl, and Mose Rossi
45. Immunological Investigations on Antarctic Fish Parasitism by Nematodes
Maria Rosaria Coscia and Umberto Oreste

171

177

181

189
193

197

201
205

209
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Contents

XV

46. The Identification and Characterisation of Graci!aria gracilis Defence Genes
Expressed in Response to a Bacterial Infection . . . . . . . . . . . . . . . . . . . . . .
Ann E. Jaffray and Vernon E. Coyne
47. Improving Enzyme Thermostability: The Thermococcus litoralis Glutamate
Dehydrogenase Model . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Costantino Vetriani, Dennis L. Maeder, Nicola J. Tolliday, Horst H. Klump,
Kitty S. P. Yip, David W. Rice, and Frank T. Robb
48. Ligand-Activated Ca2+ Channels in the Nuclear Envelope of Starfish Oocytes
Luigia Santella and Keiichiro Kyozuka

217

221

227

Section 7: Biodiversity, Environmental Adaptation, and Evolution
49. Intron as a Source of Genetic Polymorphism for Fish Population Genetics
Seinen Chow

231

50. Polymorphism of Digestive Enzymes Coding Sequences in the Crustacea
Penaeus vannamei (Crustacea Decapoda) . . . . . . . . . . . . . . . . . . . . . . . . . .
D. Sellos, C. Le Boulay, B. Klein, I. Cancre, and A. VanWormhoudt

235

51. Mating Dynamics ofthe Snow Crab (Chionoecetes opilio, Brachyura: Majidae):

An Analysis Using DNA Microsatellite Markers . . . . . . . . . . . . . . . . . . . . .
N. Urbani, B. Sainte-Marie, J.-M. Sevigny, D. Zadwomy, and U. Kuhnlein

241

52. Denaturation of Algal Phycobiliproteins Can Be Used as a Thermal Process
Indicator . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
A. Orta-Ramirez, D. M. Smith, and J. E. Merrill

245

·53. Stress Responsive Gene for UV-A in Marine Cyanobacterium
Oscillatoria sp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Tadashi Matsunaga and Akira Yamazawa

251

54. Analysis of Stress Responsive Gene for Salinity in a Marine Cyanobacterium
Synechococcus sp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Haruko Takeyama and Hideki Nakayama

255

55. Mussels Mytilus as Model Organisms in Marine Biotechnology: Polypeptide
Markers of Development and Sexual Differentiation of the Reproductive
System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Alexander T. Mikhailov, Mario Torrado, and Josefina Mendez
56. Molecular Ecology of Marine Invasions: Two Case Studies . . . . . . . . . . . . . . . . .
Jonathan B. Geller
57. A Super Heat-Stable Extracellular Proteinase from the Hyperthermophilic

Archaeon Aeropyrum pernix Kl . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
P. Chavez C., Y. Sako, and A. Uchida

259

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Contents

Section 8: Biomarkers, Symbiosis, and Viruses
58. Mannose Adhesin-Glycan Interactions in the Eup1ymna sea/opes-Vibrio
.fischeri Symbiosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
M. McFall-Ngai, C. Brennan, V. Weis, and L. Lamarcq

273

59. Temporal Control of lux Gene Expression in the Symbiosis between Vibrio
fischeri and Its Squid Host . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Karen L. Visick and Edward G. Ruby

277

60. Bacterial Symbionts of the Bryostatin-Producing Bryozoan Bugula neritina
Margo G. Haygood and Seana K. Davidson
61. Are Gamma Proteobacteria the Predominant Symbionts in the Squid

Loligo pealei? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Elena Barbieri, Deborah Hughes, Rebecca Ericson, and Andreas Teske
62. Molecular Detection of Aquatic Birnaviruses from Marine Fish and Shellfish
Satoru Suzuki

281

285
291

63. A SDS/Page/Western Blot/EIA Protocol for the Specific Detection of Shrimp
Viral Pathogens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Philip C. Loh, Lourdes M. Tapay, E. Cesar, B. Nadala, Jr., and Yuanan Lu

295

64. Expression of Capsid Proteins from Infectious Pancreatic Necrosis Virus (IPNV)
in the Marine Bacterium Vibrio anguillarum . . . . . . . . . . . . . . . . . . . . . . . .
John T. Singer, Jacqueline H. Edgar, and Bruce L. Nicholson

303

65. Detection ofCulturable and Non-Culturable Vibrio cholerae 01 in Mexico.....
Marcial Leonardo Lizarraga-Partida, Irma Wong-Chang,
Guadalupe Barrera-Escorcia, Alfonso, and V Botello
66. Molecular Characterization of Metallothionein- and Cytochrome
P4501A-CDNAS of Sparus aurata and Their Use for Detecting Pollution
along the Mediterranean Coast of Israel . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Moshe Tom, Ophira Moran, Edward Jakubov, Benzion Cavari, and
Baruch Rinkevich

307

311

Section 9: Workshops
67. Workshop on Fatty Acid Production and Metabolism: Synthetic Report
S. A. Poulet and K. Yazawa

315

68. Workshop on Biodiversity: Synthetic Report . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
J. Frederick Grassle and Jack B. L. Matthews

317

69. Workshop on Policy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Bernardino Fantini and Fernando Quezada

321

Contributors

325

Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

339

1

BIOTECHNOLOGY:
BIOLOGY OR TECHNOLOGY?
Keynote Lecture

Arthur Kornberg

INTRODUCTION TO DR. ARTHUR KORNBERG
We are privileged to open this conference by one of our most distinguished contributors to science and a spokesman for science, Dr. Arthur Kornberg. Following a brilliant
career in Biotechnology at NIH and Washington University in St. Louis, he undertook a
study of the mechanism of DNA replication which attracted international attention and led
to his Nobel Prize. Moving to Stanford University, he established one of the most outstanding Departments in Molecular Biochemistry. Their graduates and Post Doctoral Fellows are found worldwide in major research universities. After retiring as chair, Dr.
Kornberg continued to provide leadership to the scientific community, guidance to government and interpretations about science to the public. A strong supporter of the need for
basic research, his vision on how this is accomplished and how this is translated to solve
practical problems has been widely received. Recognizing this, and the interdisciplinary
nature of Marine Biotechnology, it was Nino Salvatore's wish to have Prof. Kornberg
open IMBCC'97 with a keynote address "Biotechnology: Biology or Technology?"
H.O.H.

KEYNOTE LECTURE
During this 20th century with its succession of microbe hunters, vitamin, enzyme
and gene hunters, and golden ages in medical science, the current age of gene hunting is
undeniably the most golden. We have an inexhaustible supply of genes and simple and
efficient techniques to track and capture them. We are participating in the most revolutionary advance in the history of biological and medical science. The term revolutionary is
generally overused, but not here. The effects of this advance on medicine, agriculture,
industry, and basic science have not been exaggerated.
New Developments in Marine Biotechnology, edited by LeGal and Halvorson.
Plenum Press, New York, 1998.

2

A. Kornberg

Yet even more revolutionary but generally unnoticed, is a development which, lacks
a name, has no obvious applications, but will surely lead to even more remarkable and
unanticipated practical applications. I refer to the coalescence, the confluence and the
merging of the numerous basic biologic and medical sciences into a single, unified discipline which has emerged because it is expressed in a single universal language, the language of chemistry.
The breakthrough of recombinant DNA and genetic engineering was based on the
discoveries of enzymes that make, break and seal DNA. All these basic advances were
made in academic laboratories built and supported almost entirely by funds from the NIH.
For thirty years, my research on the biosynthesis of the building blocks of nucleic acids,
their assembly in DNA replication and the training of well over a hundred young scientists, was funded with many millions of dollars without any promise or expectation that
this research would lead to marketable products or procedures. No industrial organization
had, or would ever have, the resources or disposition to invest in such long-range, apparently impractical programs. We carried out these studies to satisfy a need to understand
basic processes in cellular function. Yet to my great pleasure, such studies of the replication, repair and rearrangements of DNA have had many practical benefits.
The pathways of assembling DNA from its building blocks have been the basis for
the design of most drugs used today in the chemotherapy of cancer, AIDS, Herpes and
autoimmune diseases. These studies are also crucial to understanding the repair of DNA,
so important in the aging process, for understanding mutations and the origin of some cancers. It may seem unreasonable and impractical, call it counterintuitive, even to scientists
to solve an urgent problem, such as a disease, by pursuing apparently unrelated questions
in basic biology or chemistry. Yet, the pursuit of understanding the basic facts of nature
has proven throughout the history of medical science to be the most practical, the most
cost-effective route to successful drugs and devices. Investigations that seemed totally
irrelevant to any practical objective have yielded most of the major discoveries of medicine--X-rays, penicillin, polio vaccine, recombinant DNA and genetic engineering. All
these discoveries have come from the pursuit of questions in physics, chemistry and biology, unrelated at the outset to a specific medical or practical problem.
With regard to industrial inventions, there is a common saying: "Necessity is the
mother of invention." Not true! Rather, the reverse has proven to be true: invention is the
mother of our necessities. Inventions only later become necessities!

Quite clearly, even industrial inventions emerge from a creative process. As such
they are haphazard rather than goal-oriented. The lessons to be learned from this history
should be crystal clear. It is crucial for a society, a culture, a company, a university, to understand the nature of the creative process and to provide for its support. No matter how
counter-intuitive it may seem, basic research is the lifeline of practical advances in medicine; pioneering inventions are the source of industrial strength. The future is not predicted it is invented.
Of course it is important that basic discoveries be promptly and wisely applied to
solve practical problems. The recent applications of biotechnology to medicine have given
us major insights into diabetes, cancer and other metabolic diseases. Will these approaches
and techniques be equally effective when applied to the human brain and behavior? I am
sure they will. That human behavior is a matter of chemistry and neurons is hard for some
to accept, including physicians. We must remind them that the same chemical language
that describes the functions of the heart and liver surely applies to the basic operations of
the brain.


Keynote Lecture

3

The overriding issue in biomedical science, as I see it, is how to give our abundant
scientific talent the resources to exploit the extraordinary new technologies to advance
knowledge. Currently, a pervasive mood among productive biomedical scientists makes
them fear for continued grant support, persuades them to choose safe and practical projects over the untried and adventurous, and tempts their interest in commercial ventures.
This is clearly a state that discourages young people from entering science and drives others to abandon science for business, law and other pursuits.
The independence of an American scientist to initiate and pursue his own research
program in the biomedical sciences has been achieved because the NIH awarded research
grants to the individual scientist who is not indebted to a department head, a dean or to
university politics. The university has no choice but to give the scientist independence in
order to compete for them, for their teaching contributions and the very considerable income from indirect costs attached to their grants. Yet, it should be said, that the very competition for grantees is an essential ingredient of the success of the NIH grants program. It
depends on the fact that the private and public universities are free from centralized government controls, something virtually unique in the United States.
The expansion of research grants in many countries is highly laudable, but unfortunately the old mechanisms often prevail. In Japan, a very large sum is awarded to "centers of excellence" in which the director can exert authority over what is done and who

does it. In Europe, research programs, especially in the smaller countries, rely on grants
from the European Union. The EU requires that investigators from three or more countries find a consensus project that can be parcelled up among them. This leaves no room
for a scientist to do something utterly original and unpopular, and much time is wasted
on bureaucratic maneuverings. Recent reports indicate that in the United Kingdom the
Medical Research Council is planning to consolidate grants along similar lines. Here in
Italy, the powerful baronial organization of research granting agencies perpetuates fragmentation and favoritism.
Another problem I want to consider includes conflicts within our science, conflicts
that can reduce our effectiveness: these include conflicts between the cultures of chemistry and biology, confusion in biotechnology between biology and technology, and big science versus little science. In each of these conflicts, philosophical differences are overlaid
by strong economic, social and political forces.
First is the rift for more than a century between the cultures of chemistry and biology.
The emergence of biochemistry early in this century might have bridged the gap between
chemistry and biology but it didn't. Nor has the recent popularity of genetic chemistry.
Chemists continue to synthesize and analyze small molecules with ever greater precision, but they neglect the biological macromolecules: the proteins, nucleic acids and
polysaccharides; these seem to them too complex or too mundane. Too few chemists exploit the borderland, in which they can find rich harvests in the vast and awesome biological chemistry evolved for over a billion years.
Biologists for their part avoid enzymology. To them, enzymes are faceless components of kits or gene products inferred from sequences recognized by motifs and homologies. Biologists are so enthralled by the mysteries of evolution, development, aging and
diseases, that they resist reductionist chemical approaches and focus instead on the vital
phenomena they create by altering the genomes of their favorite organisms.
Biologists should realize that the history of science is littered with vitalistic pronouncements that reduction has gone as far as it can go. With the application of chemical
techniques of ever-increasing sensitivity and precision, they can gain a deeper and 4-dimensional understanding of biologic events.


4

A. Kornberg

Another conflict is found in the increasing influence of biotechnology enterprises.
Genetic engineering and associated technologies have been enormously successful. Yet we
must be aware that the very term biotechnology, adopted as a euphemism for genetic engineering, may blur the important distinction between biology, a quest for knowledge, as
opposed to technology, the application of that knowledge for products and profit.
Scientists and academic institutions involved in biotech enterprises are likely to be

distracted from their central mission: the pursuit of the basic understanding of nature. I am
especially concerned with another problem. There is an illusion created by the financial
and research successes of a few biotech ventures that a significant fraction of basic advances can be supplied by industry. Although such achievements are laudable, they represent only a tiny fraction, perhaps five percent of the basic knowledge needed to combat
diseases, advances which can come only from the massive federal support of research and
training.
Lastly, I want to mention still another conflict, the conflict between big and little science. Of course, we need both. There are projects that require large and expensive equipment and several disciplines to use it effectively. My concern is that with the worldwide
expansion of big science, little science will vanish. As I view the steady growth of collective science and big science, the greatest danger I see is a dampening of individual creativity and reversion to the old politics-the inevitable local politics that infects every group
and institution.
I want to recognize what deserves the most emphasis and what unites us all. It is our
unconflicted and overriding devotion to the culture of science. We must make it clear to
the public that science is great, although scientists are still people. As people, they are no
different from others: dentists, lawyers, artists, businessmen. Scientists are just as prey to
the human failings of arrogance, greed, dishonesty and psychopathy. What does set them
apart from others is the discipline of science, a practice that demands exact and objective
descriptions of progress, evidence that can be verified or denied by others.
It is the discipline of science that enables all of us ordinary people, whether we be
chemists, biologists or physicians, to go about doing the ordinary things, which, when assembled, reveal the extraordinary intricacies and awesome beauties of nature. Science not
only permits us to contribute to grand enterprises, but also offers a changing and endless
frontier for exploration.
This frontier for exploration has given me an opportunity to probe an utterly new
area after having worked on DNA for 40 years. A few years ago, I described my fascination with another polymer, one which was surely on earth before nucleic acids and proteins. It was likely a precursor and catalyst in the synthesis of RNA, DNA and proteins
and is now conserved in every bacterial, fungal, plant and animal cell. It is a chain of hundreds of phosphates linked by the high-energy bonds found in ATP. Because of the antiquity of polyP and its apparent lack of any functions, it was dubbed a "molecular fossil."
My mission has been to restore the fossil to life. We have discovered many functions of
poly P, the most intriguing is that in E. coli it is essential for the elaborate adaptations that
the organism makes for its survival after exponential growth. Mutants lacking poly P die
off quickly. Simply put, poly P is essential for graceful aging in E. coli. The enzyme that
makes polyP in bacteria is highly conserved. These include M. tuberculosis, Helicobacter
pylori, Neisseria meningitidis and other pathogens, and also cyanobacteria and streptomyces. We are working with medical microbiologists to determine the influence of poly P on
the virulence of these pathogens and the production of antibiotics in Streptomyces.
People wonder whether the computer revolution and other advanced technologies

have altered the way we do bioscience research. Can our research now be engineered and


Keynote Lecture

5

pursued by formula? Not yet. The technical tools are indispensable, but the practice of science remains in essence highly creative and its province is Nature. Sir Karl Popper, an
eminent philosopher of science and society, who died three years ago in London, said that
"next to music and art, science is the greatest, most beautiful and most enlightening
achievement of the human spirit." I would place science first.
We probe the inexhaustible mysteries of Nature from a variety of directions, and
with different intensities and styles. These probings are determined by our emotions, our
moods and our cultural heritage, much as these influence tne artist. The major discoveries
in bioscience are more often intuitive or serendipitous than the result of logical analysis.
The machines we use produce images and compositions of objective precision. But
this should not obscure the fact that we use these machines as tools, with tastes as distinctive as those that painters use their palettes, composers their notes, and authors their words
in creating their images of Nature. Seneca, the great Roman statesman and philosopher,
once said: "All art is but imitation of Nature." What we try to do in science is to get ever
closer to Nature. In the art of medicine, we try to find for the individual a harmonious
niche in Nature.

REFERENCE
FASEBJournal, 1997,11:1209-1214


2

THE PARADOX OF GROWTH
ACCELERATION IN FISH

Jose de la Fuente,* Isabel Guillen, and Mario P. Estrada
Mammalian Cell Genetics Division
Centro de Ingenieria Genetica y Biotecnologia
P. 0. Box 6162, Havana, Cuba

1. INTRODUCTION
Growth is a complex and tightly regulated process in fish. The growth hormone
(GH) is a polypeptide playing a key role in the process of growth and is synthesized
mainly by somatotrophos in the anterior pituitary gland. Release of GH from the pituitary
gland is thought to be controlled primarily by hypothalamic factors. Once in the circulation, a substantial proportion of the GH appears to bind to a specific binding protein, probably responsible for the control of the hormone half life in the circulation. After binding to
specific cell receptors, GH stimulates, primarily in the liver, Insulin-like growth factor
(IGF-1 and IGF-11) synthesis and secretion to elicit the growth promoting action in an
autocrine and paracrine fashions. IGF also elicits a negative feedback on the secretion of
GH in the pituitary gland in tilapia (Guillen et al., in press).
Growth acceleration has been reported for tilapia and other fish species. However,
these results did not address the question regarding the levels of ectopic GH required to
achieve maximal growth acceleration without causing detrimental effects to the animals.
This is a fundamental question to understand the process of growth in fish and to effectively manipulate this process.

2. EXPERIMENTALPROCEDURES
The details of the experiments considered here have been published elsewhere
(Guillen et al., 1996; Hernandez et al., 1997).
• E-mail:)
New Developments in Marine Biotechnology, edited by Le Gal and Halvorson.
Plenum Press, New York, 1998.

7


8

J. De La Fuente et al.

3. RESULTS AND DISCUSSION
3.1. The Administration of High Doses of Recombinant tiGH Results in
Growth Inhibition in Tilapia
Cloned eDNA for tilapia GH (tiGH) was expressed in E. coli. After purification,
recombinant tiGH was correctly folded and biologically active. The growth of juvenile
tilapia (0. hornorum) was followed after intraperitoneal injections of recombinant tiGH
(0, 0.1, 0.5 and 2.5 J.lg/g body weight (gbw); 13 tilapia per group) at intervals of 7 days.
The control group received injections of vehicle plus 5 J.lg BSA/gbw. The level of growth
acceleration (in %) was determined at the end of the experiment by comparing the mean
weight of tilapia in the experimental groups with the control tilapia. A Student t-Test was
used to compare the results. In the week 9 of the experiment, the group receiving 0.5 J.lg
tiGH/gbw showed a 6% growth acceleration (p=0.05) whereas the group receiving 0.1
J.lg/gbw showed a 2% growth acceleration. In the group with the highest dose, a growth
retardation of 1% was recorded.
These results evidenced a dose-dependent effect of tiGH administration on the
growth performance of juvenile tilapia at the doses of 0.1 and 0.5 J.lg tiGH/gbw. However,
the injection of 2.5 J.lg tiGH/gbw produced a negative effect on the growth performance.

3.2. Low Level Ectopic Expression of tiGH Results in Growth
Acceleration in Transgenic Tilapia
To assay the effect of different expression patterns of ectopic tiGH, 4 lines of transgenic tilapia (0. hornorum) were generated with chimeric constructs containing the tiGH
eDNA, 5' regulatory sequences derived from the human cytomegalovirus (CMV) or Rous
sarcoma virus (RSV), polyadenylation sites from the SV40 and the first intron from the
trout GH gene (INT) (de la Fuente et al., 1995; Hernandez et al., 1997; Table 1).
Table 1. Summary of the characterization of transgenic tilapia lines
Tilapia lines•
Fl (PIx wt)

F2(FI X Fl)
FT"
F2•'•
[Fl (PI x wt)]
RSV>+INT>tiGH>SV40
[FI (PI x wt)]
CMV>-INT>tiGH>SV40
[FI (PI x wt)]
CMV>+INT>tiGH>SV40
Non-transgenic control siblings

CMV>tiGH>CAT>SV40

tiGHRNA
levelsb

tiGH protein
levels•

Growth
accelerationd

References'

5
+
+
+
240
23

8
0

10
+
+
+
78
723
60
0

82% (p=O.OOI)
55% (p=0.009)
62% (p=O.OOS)
31% (p=0.07)
0%
0%
3.4% (p=0,006)

1,2
3
3
3
1,4
1,4
1,4

•Hybrids (predominantly 0. hornorum although the breeding history is not known). Wt, wild type hybrid 0. homorum
tilapia; -/+, heterozygous; +/+, homozygous.

bRNA levels (in arbitrary units) were calculated by summarizing the results of Northern blot analyses in the liver, gonads
and muscle. Signals in the X-ray films were scanned and normalyzed against gliceraldehyde 3 phosphate dehydrogenase.
+,denotes presence oftiGH RNA in muscle samples analyzed by in situ hybridization.
'Tilapia ectopic GH protein levels (in arbitrary units) were calculated by summarizing the exposure time required for photography (employing an Olympus exposure control unit) in gonad, heart and muscle tissue sections after immunohistochemical analysis with anti-tiGH-anti rabbit IgG-Rhodamine conjugate. Values were normalized against the control. +,
denotes presence oftiGH in non-quantitated tissue sections.
dGrowth acceleration in transgenic tilapia compared to non-transgenic siblings (Student t-Test).
c I, de Ia Fuente et ai.. I 995; 2, Martinez et al., 1996; 3, Guillen et al., 1996; 4, Hermindez et al., 1997.


The Paradox of Growth Acceleration in Fish

9

Different patterns of ectopic expression of tiGH were detected in gonad, liver, brain,
heart and muscle cells of transgenic tilapia lines by RNA and/or protein analysis (Table I;
Guillen et a!. 1996; Hernandez et a!., 1997). Transgenic lines with lower ectopic tiGH
mRNA levels were the only showing growth acceleration. Small variations in the tiGH
levels resulted in big changes in the level of growth acceleration, suggesting that the
ectopic expression of tiGH promoted growth only at low expression levels (Table 1), a
fact that was also noticed in the experiment described before injecting different doses of
recombinant tiGH . Furthermore, 4 month old transgenic homozygous (F2+1+) and heterozygous (FT 1+) tilapia and non-transgenic siblings were studied for 3 months grown communally in the same pond (Guillen et a!., 1996). The results suggested a transgene-dosage
effect (Table I).
Groups working with relatively weak promoters have reported growth acceleration
in transgenic salmon (Devlin et a!., 1994) and carp (Zhu, 1992) while reports employing
the strong RSV promoter in transgenic carps showed only modest levels of growth acceleration (Zhang et a!., 1990). Furthermore, Zhang et a!. ( 1990) reported that transgenic
common carp carrying RSV>rtGHlcDNA and expressing the transgene at low levels grew
faster than those containing higher rtGH levels. These studies suggested that high levels of
GH may produce inhibitory effect on growth (Zhang eta!., 1990; Lu et a!., 1992). These
results are in accordance with those reported here for transgenic tilapia.

3.3. A Model for Growth Acceleration in Tilapia
The results obtained by us resulted in a paradox: high exogenous GH levels did not
promote growth, but rather could produce a growth retardation effect. However, low levels
of exogenous GH result in growth acceleration. This "exogenous growth hormone to
accelerate growth in fish paradox" remains the "French red wine paradox"; non is as negative as too much, but little could be beneficial.
The effect of ectopic tiGH levels over a certain value resembled in tilapia the physiological situation of low condition factor (because, for example, of low food availability;
Sumpter eta!., 1992; Guillen eta!., in press). It has been reported that fish with a low condition factor have elevated GH plasma concentrations and low levels of IGF-I, factors that

GROWIH RETARDATION
(no receptor dlmertzatlon and,
therefore, no signal transduction)
GROwn! ACCELERATION
(better receptor occupancy)
Figure I. A model for our hypothesis on the effect on growth of ectopic GH in tilapia. Elevated GH levels could
down-regulate the signal-transducing capacity ofGH and/or IGF receptors, thereby not accelerating growth. Low
ectopic GH levels could accelerate growth by permitting a better receptor occupancy, thus optimizing growthpromoting activity.


10

J. De La Fuente et al.

result in growth retardation (Sumpter, 1992; Guillen et a!. in press). In the two transgenic
lines with higher ectopic tiGH mRNA levels no growth acceleration was recorded (Table 1).
These results conduced as to the hypothesis that elevated GH levels could downregulate the level, or more likely the signal-transducing capacity, ofGH and/or IGF receptors, thereby not accelerating growth. An excess in GH circulating levels could prevent the
formation of the active GH-receptor complex by inhibiting the necessary for biological
activity dimerization reported for the human GH receptor (Wells, 1996). Low ectopic GH
levels could accelerate growth by permitting a better receptor occupancy, thus optimizing
growth-promoting activity. This hypothesis resulted in the model depicted in Figure 1.
However, alternative pathways may be also affected with the ectopic expression of GH,

affecting the process of growth in fish.

ACKNOWL EDGMENTS
We would like to thank colleagues from our group for fruitful discussions. The work
from our group was partially supported by the International Centre for Genetic Engineering and Biotechnology Collaborative Research Program (project CRP/CUB93-05).

REFERENCES
de Ia Fuente J, Martinez R, Estrada MP, Hernandez 0, Cabrera E, Garcia del Barco D, Lleonart R, Pimentel R,
Morales R, Herrera F, Morales A, Guillen 1., Piria JC ( 1995) Towards growth manipulation in tilapia (Oreochromis sp.): generation of transgenic tilapia with chimeric constructs containing the tilapia growth hormone eDNA. J. of Marine Biotechnology 3:216--219.
Devlin RH, Yesaki TY, Biagi CA. Donaldson EM. ( 1994). Extraordinary salmon growth. Nature 371: 209--210.
Guillen I, Martinez R, Hernandez 0, Estrada MP, Pimentel R, Herrera F, Morales A, Rodriguez A, Sanchez V,
A bad Z, Hidalgo Y, Lleonart R, Cruz A, Vazquez J, Sanchez T. Figueroa J, KrauskopfM and de Ia Fuente J
( 1996) Aquaculture Biotechnology Symposium Proceedings.Edited by Edward M. Donaldson and Don
D.MacKinlay.lntern ational Congress on the Biology of Fishes. San Francisco State University July 14-18,
pp. 63-72.
Guillen I, Estrada MP, Morales R. Melamed P and de Ia Fuente J. ( 1997). The interrelation of body growth with
growth hormone, insulin-like growth factor and prolactin levels in juvenile tilapia (Oreochromis aureus).
Minerva Biotecnologica (in press).
Hernandez 0, Guillen 1., Estrada MP, Cabrera E, Pimentel R., Piiia JC. Abad Z, Sanchez V, Hidalgo Y, Martinez
R., Lleonart R. de Ia Fuente J ( 1997) Characterization of transgenic til apia lines with different ectopic expression oftilapia growth hormone. Molecular Marine Biology and Biotechnology (in press).
Lu, JK. Chen T.T, Chrisman C.L., Andrisani OM. Dixon JE. ( 1992) Integration, expression and germ-line transmission of foreign growth hormone genes in medaka (01yzias latipes). Molecular Marine Biology and
Biotechnology 1(4/5): 366--375.
Martinez R, Estrada MP, Berlanga J, Guillen 1., Hernandez 0, Cabrera, E, Pimentel R., Morales R, Herrera F, Morales, A, Piiia. JC, A bad Z, Sanchez V, Melamed P, Lleonart R. de Ia Fuente, J ( 1996) Growth enhancement
in transgenic tilapia by ectopic expression of tilapia growth hormone. Molecular Marine Biology and
Biotechnology 5( 1): 62-70.
Sumpter J.P. ( 1992) Control of growth of rainbow trout (Oncorhynchus mykiss). Aquaculture I 00: 299-320.
Wells JA ( 1996) Binding in the growth hormone receptor complex. Proc Natl Acad Sci USA 93: 1--6
Zhang P, Hayat M, Joyce C, Gonzalez-Villaseno r LJ, Lin CM, Dunham R, Chen TT, Powers, DA (1990) Gene
transfer, expression and inheritance of pRSV-rainbow trout GH-cDNA in the common carp, O,prinus car-

pio (Linnaeus). Mol.Repro.Dev. 25: 3-13.
Zhu Z ( 1992) Generation of fast growing transgenic fish: Methods and Mechanisms. In: Transgenic Fish. Hew,
C.L. and Fletcher, G.L. (eds.). World Scientific Publishing Co., Singapore, pp. 92-119.


3

GENE TRANSFER IN ZEBRAFISH ENHANCED
BY NUCLEAR LOCALIZATION SIGNALS
Philippe Collas and Peter Alestrom*
Department of Biochemistry
Norwegian College of Veterinary Medicine
P.O. Box 8146 Dep., N-0033 Oslo, Norway

1. INTRODUCTION
Transgenic fish are routinely produced by injection of plasmid DNA into eggs (reviewed by Flechter and Davies, 1991 ). In zebrafish, the pronuclei of fertilized eggs are not
visible, thus the DNA is injected into the cytoplasm or the yolk. Cytoplasmically injected
DNA can integrate into the genome and be transmitted through the germline (Stuart et al.,
1988; 1990). Frequencies of trans gene integration and germ1ine transmission may be as high
as 25% (Stuart et al., 1988; Culp et al., 1991) but are often in the order of a few percents.
The low frequency of transmission of a trans gene to F 1 generation in zebrafish may
be explained by factors controlling DNA stability, nuclear import and chromosome integration. Transgenes are often degraded or rearranged (Iyengar and MacLean, 1995), often remain extrachromosomal (Stuart et al., 1990), and when integrated, may be transcriptionally
silent (Culp et al., 1991 ). Since the DNA is injected into the egg cytoplasm, rapid embryonic
cell divisions may favor transgene integration at later stages of development, leading to a
high degree of mosaicism (Flechter and Davies, 1991 ). To achieve nuclear uptake and chromosome integration of the DNA, high numbers (> 106 ) of vectors are generally injected.
This results in high concentrations of foreign DNA within the embryo, leading to increased
risks of toxicity (Flechter and Davies, 1991 ).
Several methods have been developed to improve the efficiency of transgene integration into the host genome. They include (1) the use of pseudotyped viruses (zebra fish;
Lin et al., 1994), (2) trans gene integration mediated by a retroviral integrase protein (zebrafish; Ivies et al., 1993), (3) integration by transposable elements (Drosophila; Kaufman
and Rio, 1991 ), and (4) binding of DNA to nuclear proteins (mammalian cells; Wienhues

et al., 1987) or nuclear localization signals (zebrafish; Collas et al., l996a; Collas and
• *Author for correspondence. Phone: +47 22 96 45 71; Fax: +47 22 60 09 85
New Developments in Marine Biotechnology, edited by LeGal and Halvorson.
Plenum Press, New York, 1998.

11