Gene families : studies of DNA, RNA, enzymes and proteins : proceedings of the October 5-10, 1999 congress, Beijing, China, the 10th International Congress on Isozymes 9789812810557, 9812810552

Table of contents :
Clement Markert (G L Hammond)
Identification of Novel Gene Family Members Based on Efficient Full-Length cDNA Cloning (J Gu et al.)
Aldehyde Dehydrogenases of Human Corneal and Lens Epithelial Cells (R S Holmes)
X-Chromosome Inactivation During Spermatogenesis: The Original Dosage Compensation Mechanism in Mammals? (J R Mc Carrey)
Probing for the Basis of the Low Activity of the Oriental Variant of Liver Mitochondrial Aldehyde Dehydrogenase (B Wei & H Weiner)
The Roles of Carbonic Anhydrase Isozymes in Cancer (W R Chegwidden et al.)
MHC Class II Suppression by Trophoblast cDNAs (G L Hammond et al.)
Molecular Information Fusion for Metabolic Networks (R Hofestadt et al.)
Effect of Heterogeneous Sperm and Hybridization of DNA Fragment in Allogynogenetic Silver Crucian Carp (D Xia et al.)
Gene Expression During Carrot Somatic Embryogenesis (N Wu)
and other papers.

Citation preview

GENE FAMILIES Studies of DNA, RNA, Enzymes and Proteins

f^

Editors ,iong Xue, Yongbiao Xue, Zhihong Xu, Roger Holmes, Graeme L Hammond & Hwa A. Lim

World Scientific

GENE FAMILIES Studies of DNA, RNA, Enzymes and Proteins

Published by World Scientific Publishing Co. Pte. Ltd. P O Box 128, Farrer Road, Singapore 912805 USA office: Suite IB, 1060 Main Street, River Edge, NJ 07661 UK office: 57 Shelton Street, Covent Garden, London WC2H 9HE

British Library Cataloguing-in-Publication Data A catalogue record for this book is available from the British Library.

GENE FAMILIES: STUDIES OF DNA, RNA, ENZYMES AND PROTEINS Copyright © 2001 by World Scientific Publishing Co. Pte. Ltd. All rights reserved. This book, or parts thereof, may not be reproduced in any form or by any means, electronic or mechanical, including photocopying, recording or any information storage and retrieval system now known or to be invented, without written permission from the Publisher.

For photocopying of material in this volume, please pay a copying fee through the Copyright Clearance Center, Inc., 222 Rosewood Drive, Danvers, MA 01923, USA. In this case permission to photocopy is not required from the publisher.

ISBN 981-02-4384-7

Printed in Singapore by World Scientific Printers

GENE FAMILIES Studies of DNA, RNA, Enzymes and Proteins Proceedings of the October 5 - 1 0 , 1 9 9 9 Congress, Beijing, China The 10th International Congress on Isozymes

Editors

Guoxiong Xue Institute of Developmental Biology Chinese Academy of Sciences, Beijing, China

Yongbiao Xue, Ph.D. Institute of Developmental Biology Chinese Academy of Sciences, Beijing, China

Zhihong Xu, Ph.D. Peking University, Beijing, China

Roger Holmes, D.SC. University of Newcastle, New South Wales, Australia

Graeme L. Hammond, M.D. Yale University, School of Medicine, Connecticut, USA

Hwa A. Lim, Ph.D., MBA D'Trends Inc., California, USA

V f e World Scientific w l

Singapore • New Jersey'London • Hong Kong

Honorary President: Clement L. Markert Chair: Zhihong Xu Co-Chairs: Boqing Qiang Fangzhen Sun Guoxiong Xue (Executive) Laining Yu

International Executive Committee: Carla Frova (Italy) Erwin Goldberg (USA) Roger Holmes (Australia) Hans Jornvall (Sweden)

Masataki Mori (Japan) C. Schnarrenberger (Germany) John VandeBerg (USA) Guoxiong Xue (China)

National Advisory Committee: Songlin Chen Yongfu Chen Zhu Chen Miao Du Guofan Hong Yunde Hou Jifang Huang Kam-Len D. Lee Zhensheng Li Cheng Ma Bo Tian Guihai Wang Guanhua Xu Yongbiao Xue Longfei Yan Shaoyi Yan Qifa Zhang Rongquan Zhang Lihuang Zhu Zuoyan Zhu

International Advisory Committee Atonnio Blanco (Argentina) W. Richard Chegwidden (USA) Jacques Drouin (Canada) Clara Gozodezki (Mexico) Hwa A. Lim (USA) Jose Luis Millan (USA) Atsushi Nakazawa (Japan) Eviatar Nevo (Israel) P.R.K. Reddy (India) Francisco Salzano (Brazil) Maria de Fatima L. Santos (Portugal) John Scandalios (USA) Wolfgang Scheffrahn (Switzerland) Michael J. Siciliano (USA) Oleg Serov (Russia) Y.H. Tan (Singapore) Athanasios Tsaftaris (Greece) Jerry Wang (Canada) Diter von Wettstein (USA) Suren Zakian (Russia)

Sessions and Session Chairpersons: (In chronological order) Plenary Symposium:

John VandeBerg Gerald Stranzinger Erwin Goldberg Che-Kun Shen Roger Holmes Boqing Qiang Yongbiao Xue

Gene Families and Isozymes:

Desmond Cooper Henry Weiner Richard Chegwidden

Population Variation of Gene Families:

Robert Gracy

Gene Structure and Mapping:

Jiayang Li

Gene Families and Human Diseases:

Hans Jornvall

Gene Families and Evolution:

Eviata Nevo Shusen Liu

Genetic Mutations and Diseases:

Masami Muramatsu

Genomes and Bioinformatics:

Hwa A. Lim Cheng Jing

Gene Families and Plants:

Desmond Cooper Claus Schnarrenberger

Gene Families and Gene Expression:

Jacques Drouin John R. McCarrey Frederick Sweet

Mammalian Gene Families:

Yongfu Chen

Gene Families and Biotechnology:

Zuoyan Zhu P.R.K. Reddy

Young Scientists:

Fuchu He Fangzhen Sun Congress Liaison: Wang Ning

ACKNOWLEDGEMENTS Technical & Logistics Support for Proceedings Production DTrends, Inc., USA

Financial Support for Proceedings Production DTrends, Inc., USA

MS WORD Editor Hwa A. Lim [email protected]

Manuscript Review Committee Charles H. Blomquist (Health Partners Ramsey Clinic, Minnesota) Xiao Zhuo Chen (Ohio University, Ohio) Paolo Fortina (University of Pennsylvania School of Medicine, Pennsylvania) Erwin Goldberg (Northwestern University, Illinois) Robert Gracy (University of North Texas Health Science Center, Texas) Perry B. Hackett (University of Minnesota, Minnesota) Graeme Hammond (Yale University School of Medicine, Connecticut) Roger Holmes (University of Newcastle, Australia) Larry Kricka (University of Pennsylvania, Pennsylvania) Hwa A. Lim (DTrends, Inc., California) John R. McCarrey (Southwest Biomedical Foundation, Texas) Jose Luis Millan (The Burnham Institute, California) Peter Parsons (University of La Trobe, Australia) Tim Robbins (University of Nottingham, UK) Claus Schnarrenberger (Freie Universitat Berlin, Germany) Frederick Sweet (Washington University School of Medicine, Washington) Alan R. Templeton (Washington University, Missouri) John L. VandeBerg (Southwest Biomedical Foundation, Texas) Guoshun Wang (University of Iowa College of Medicine, Iowa) Henry Weiner (Purdue University, Indiana) Ditter von Wettstein (Washington State University, Washington) Edgar Wingender (Research Group Bioinformatics, GBF, Germany)

National Sponsoring Organizations Chinese Academy of Sciences National Natural Science Foundation of China Chinese Association for Science & Technology Chinese Committee for International Union of Biological Sciences Changjiang Fisheries Institute, Chinese Fisheries Academy of Sciences Molecular Developmental Biology Open Lab, Chinese Academy of Sciences Plant Molecular Developmental Biology Lab, Institute of Developmental Biology

Industry Organization Sponsoring Congress DTrends, Inc., USA http://www.d-trends.com

Industry Exhibitors Gene Company Ltd. Amersham Pharmacia Biotech Shanghai Sangon Co. Ltd. Bio-Rad LTI Co. Promega Co. EG&G Co. Perkin-Elmer Applied Biosy stems Eppendorf Olympus Co. Nikon Co. Novo Nordisk Beijing Tianxiangren Biotechnology Co. Ltd. Beijing Liuyi Instrument Factory Pall Co. Millipore Biotinge-Tech Co. Ltd. Beijing SBS Biotechnology Inc.

PREFACE As Chairman of the International Executive Committee of the 10 International Congress on Genes, Gene Families, and Isozymes, I am pleased to make the opening remarks for this special volume. The meeting, held in Beijing, coincided with the celebrations of the 50th anniversary of the founding of People's Republic of China. The Congress was organized by Dr. Guoxiong Xue and his Co-Chairs, Drs. Boqing Qiang, Fangzhen Su, and Laining Yu. The Chair of the Congress, Dr. Zhihong Xu, is Vice President of the Chinese Academy of Sciences, and since the Congress was appointed also as President of Peking University. As Presiding Chair of the Congress, and on behalf of all of the participants, I thank the organizers and the Chair for the effort they committed to making the meeting a huge success scientifically as well as socially and culturally. The 10th Congress continued the precedent, established at the 9th Congress, of emphasizing genes and gene families as the primary determinants of isozymes and of protein multiplicity. Toward that goal, the organizers structured the Congress around the following themes: Gene Families and Isozymes Gene Families and Enzymes Population Variation of Gene Families Gene Families and Human Disease Genetic Mutation and Disease Gene Families and Evolution Mammalian Gene Families Gene Families and Plants Gene Families and Gene Expression Gene Families and Biotechnology Genomes and Bio-information The 10th Congress also continued the tradition of exploring the interfaces among various biological disciplines, rather than focusing on individual disciplines as is most common at scientific meetings. Cross-fertilization was highly evident among cognate disciplines in the biological sciences in studies of disease, evolutionary biology, medical genetics and gene regulation. Moreover, research was presented involving a wide range of organisms including bacteria, protozoa, plants, and mammals. This volume contains selected papers from the Congress, all of which have gone through a rigorous peer review process. These papers are representative of the breadth of scientific topics discussed at the meeting and of the high scientific quality of the meeting. I thank the authors, the reviewers, and the editors for their IX

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commitment to the excellence of this volume as a lasting tribute to the Congress and to the field that was founded by Professor Clement Markert. The 10* Congress began with a tribute to Professor Markert, who passed away just four days before the opening of the meeting. Professor Markert had been named as the Honorary President of the Congress, in recognition of his role in discovering isozymes and pioneering the concepts of isozymes and gene families. Professor Graeme Hammond presented a moving tribute to Professor Markert and to the professional and personal contributions that he made to science and to society throughout his life. The 10* Congress was held 30 years after Dr. Markert and his colleagues first published the concept of isozymes. The Congress had a strong international flair, with presentations from scientists representing 29 countries and regions. They included Australia, Austria, Belorussia, Brazil, Canada, the Czech Republic, Denmark, Finland, Germany, Greece, Hong Kong Special Administrative Region of China, India, Ireland, Israel, Italy, Japan, Malaysia, The Netherlands, People's Republic of China, Portugal, Russia, Singapore, Spain, Sweden, Switzerland, Taipei, Thailand, United Kingdom, and USA. As has been traditional from this series of Congresses, the organizers arranged a variety of special social and cultural activities that complemented the scientific activities of the Congress. These included a superb welcoming reception, a visit to the Chinese Opera, and a day trip to the Great Wall and other local attractions. Since their inception in 1961, this series of congresses has provided an opportunity for scientists working in a wide variety of fields involving isozymes and gene families to interact and to learn from the isozyme concept as it is applied diversely to many biological disciplines. I look forward to the 11 International Congress on Genes, Gene Families, and Isozymes to be hosted by Dr. Hans Jornvall of the Karolinska Institute in Stockholm in 2001, the 30th anniversary of this series of congresses.

September 2000 JOHN L. VANDEBERG

Director, Southwest Regional Primate Research Center San Antonio, Texas, USA E-mail: [email protected]

OBITUARY Clement L. Markert (1917-1999) The concept of isozymes was developed by Clement Markert and Freddy Moller in 1959,' which paved the way for extensive studies on enzyme, protein and gene multiplicity across all living organisms. This important scientific discovery has had a profound influence on the biological sciences for more than 40 years, and provided the basis for regular international meetings to discuss the biological and biomedical implications of enzyme multiplicity. More recently, this concept has been extended to a wide range of gene families for DNA, RNA, proteins and enzymes. As the Honorary President of the 10th International Congress on Genes, Gene Families and Isozymes, recently held in Beijing China in October 1999, Dr Markert was planning to attend and participate in his 10th 'Isozyme' Conference. Unfortunately, it was announced at the Congress Opening by Clem's friend and collaborator from Yale University, Dr Graeme Hammond, that he had passed away four days earlier following a recent illness. As Clem would have wanted it, the Congress proceeded in his absence and was an outstanding success. All of us attending the meeting, however, were saddened by the absence of a wonderful scientist who established the field of gene families and isozymes as a fundamental concept of living organisms. We also missed his friendship, good humor and contributions of criticism, advice and commendation of the papers presented at the Congress. Clem Markert was born on April 11 1917 in Los Animas, Colorado USA, and passed away in Colorado Springs on October 1 1999. His 82 years were filled with hard work, adventure, outstanding science, international travel, love for his wife Margaret and their children Alan, Robert and Betsy, and in his younger years, controversy. Clem graduated from the University of Colorado in 1940, following activities in Spain fighting with the International Brigades against the fascist regime in that country in 1938. During the Second World War, he served in the U.S. merchant navy, following completion of his Masters degree at UCLA. After the war, he completed his Ph.D. in 1948 at John Hopkins University in Baltimore, followed by a two-year Postdoctoral at the California Institute of Technology. His first academic appointment as Assistant Professor was at the University of Michigan during 195056. It was during this period that he came under scrutiny by the Committee for Unamerican Activities, commonly referred to as the McCarthy Committee. The response from Clem and Margaret, and the outstanding support provided by his

Markert and F. Moller, Multiple forms of enzymes: Tissue, ontogenetic and species specific patterns. Proc. Natl. Acad. Sci. USA 45 (1959) pp. 753-763. XI

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scientific colleagues and friends, reflected their strength and resilience, and their desire to ensure that free speech was a protected right. His appointment at Johns Hopkins University during 1956-65 was enormously productive and rich in biological discovery and conceptual development. In 1957, the 'zymogram' technique was published jointly with Robert Hunter, a colleague from the University of Michigan. This method combined the resolution power of multiple forms of enzymes by starch gel electrophoresis with the specificity derived from histochemistry in the staining of enzymes. It was applied initially to esterases and subsequently to many other enzymes, including lactate dehydrogenase. It was the latter path-breaking work that led to the 'isozyme' concept, and many scientific discoveries around the world, with studies on micro-organisms, plants and animals revealing the extensive multiplicity of genes, gene families and isozymes in all biological species. In 1965, Dr. Markert moved to Yale University to become Chairman of the Department of Biology during 1965-1971, and continued on at Yale as Professor of Biology until 1986. During 1974-86, he served as the Director of the Center for Reproductive Biology, reflecting his pioneering role in the related field of transgenics. Together with his collaborator, Jon Gordon, Clem developed a powerful tool for developmental genetics, involving the microinjection or micromanipulation of nuclei of mammalian eggs. After 21 years at Yale University, Clem and Margaret moved to North Carolina State University, where he was appointed as a Distinguished University Professor during 1986-93. There, he joined a longstanding friend and colleague, John Scandalios, who had been appointed to a similar prestigious position supported by the State of North Carolina, in its enhancement program for scientific research. At the age of 76, Clem became an Emeritus Distinguished Professor of the University, and returned to live in Colorado Springs, which is near where the Markert family spent their summer break at their 'cabin' high in the Santa Cruz Mountains. During this distinguished career, Clem was recognized with many honors and awards, including election to the National Academy of Sciences (governing Council member during 1970-71,1977-1980); American Institute of Biological Sciences (President, 1965); American Genetic Association (President, 1980); American Society of Naturalists (Vice-President, 1967); American Society of Zoologists (President, 1967); and many other societies. He has served as a scientific editor on a number of journals and other publications, including positions as Managing Editor of the Journal of Experimental Zoology (1963-85); member of the Editorial Board of the Archives of Biochemistry and Biophysics; Differentiation; Cancer Research; Developmental Genetics; Transgenics; and Editor of the Proceedings of the 3 r , and

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R.L. Hunter and C.L. Markert, Histochemical demonstration of enzymes separated by zone electrophoresis in starch gels. Science 125 (1957) pp. 1294-1295.

Obituary

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5th ^th j S 0 Z y m e Congresses and of the Prentice-Hall Series in Developmental Biology. Ten international congresses have now been held on isozymes and gene families: the first (1961) and second (1966) in New York under the sponsorship of the New York Academy of Sciences, and the third (1974) at Yale University and Chaired by Clem, with outstanding support from Margaret. Subsequent Congresses have been regularly held at various locations, including Austin, Texas (4 , 1982); Island of Kos, Greece (5th, 1986); Toyama, Japan (6th, 1989); Novosibirsk, Russia (7th, 1992); Brisbane, Australia (8th, 1995); San Antonio, Texas (9th, 1997); and Beijing, China (10th, 1999). All of these Congresses, with the exception of the last Congress, were attended by Clem, who played major roles in them all by the delivery of Plenary Lectures as Congress President or Plenary/Symposium Chairman, and strong participation in the scientific and social activities of the Congresses. We have not only lost a great scientist and pioneer in field of isozymes and gene families, he has been a friend, mentor, student and postdoctoral supervisor, collaborator, editor, referee and supportive critic for many of us. Clem has been strongly supported throughout his distinguished career by his wife Margaret, who accompanied him to conferences, Isozyme Congresses and other visits with friends and colleagues around the world. They were always generous hosts, inviting us into their homes in Ann Arbor, Baltimore, New Haven, Raleigh and Colorado Springs, as well as to their mountain retreat in the Santa Cruz mountains. Clem was a strong person in every sense of the word, physically and mentally. He was an engaging conversationalist with a remarkably broad knowledge of the biological sciences, and with strong interests in science policy, which were expressed at the highest levels in government and scientific organizations. He also was an internationalist, with a healthy cohort of 'foreigners' undertaking graduate and postdoctoral study. His contribution to the development of science in many countries is well known, including China and Russia. Clem Marker! will be remembered by many scientists, colleagues and friends around the world and has given all of us a lasting legacy in the biological sciences with the isozyme concept now being applied in a broad range of gene families in molecular biology and biochemistry, cellular differentiation, developmental biology, biomedical science, gene regulation, structure and function of enzymes and isozymes, transgenics, and population biology. Farewell Clem. Our condolences go to Margaret and the Markert family with our love and best wishes. September 2000 ROGER S. HOLMES

Vice-Chancellor and President, The University of Newcastle Callaghan, New South Wales, AUSTRALIA Email: vc@newcastle,edu.au

TABLE OF CONTENTS Preface

ix

Obituary

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Clement Markert G. L. Hammond

1

Identification of Novel Gene Family Members Based on Efficient Full-Length cDNA Cloning J. Gu, X.-Y. Wu, M. Ye, Q.-H. Zhang, Z. G. Han, H.-D. Song, Y. -D. Peng and Z. Chen Strategies for Testis Specific Gene Expression E. Goldberg Oxidized Isoforms as Diagnostic Biomarkers of Alzheimer's Disease R. W. Gracy, J. M. Talent, C. Malakowsky, R. Dawson, P. Marshall and C. C. Conrad Transgenic Fish and Biosafety W. Hu and Z. Zhu

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29

39

Aldehyde Dehydrogenases of Human Corneal and Lens Epithelial Cells R. S. Holmes

49

X-Chromosome Inactivation During Spermatogenesis: The Original Dosage Compensation Mechanism in Mammals? J. R. McCarrey

59

Molecular Evolution and Environmental-Stress E. Nevo

73

Nitric Oxide Related Enzymes and Coronary Artery Disease X. L. Wang

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xvi Pathways, Compartmentation and Gene Evolution C. Schnarrenberger and C. F. Martin

103

Tomato CF Genes for Resistance to Cladosporium fulvum C. M. Thomas, M. S. Dixon and J. D. G. Jones

115

Gene Expression and Intermolecular Forces in Estrogen/Receptor Binding Q. Chen, S. Adler and F. Sweet

133

Probing for the Basic of the Low Activity of the Oriental Variant of Liver Mitochondral Aldehyde Dehydrogenase B. Wei and H. Weiner

141

S RNases and Self and Non-self Pollen Recognition in Flowering Plants Y. Xue, H. Cui, Z. Lai, W. Ma, L. Liang, H. Yang and Y. Zhang

149

The Roles of Carbonic Anhydrase Isozymes in Cancer W. R. Chegwidden, I. M. Spencer and C. T. Supuran

157

Biochip and Miniaturization J. Zhang, W. -L. Xing, Y. -X. Zhou and J. Cheng

171

Functional Genomics: A Platform for the Discovery of New Therapies D. Cohen

179

A Novel Mathematical Analysis of Human Leukocyte Antigen (HLA) Polymorphism B. Feng, D. Pan, S. Chen, Z. Ye and A. Xu

185

Characterization of a New Tissue-Specific Mutation of the Yellow Gene Which Supports Transvection J.-L. Chen, J. Liu, K. Huisinga, P. Geyer, J. Mossis and C.-T. Wu MHC Class II Suppression by Trophoblast cDNAs G. L Hammond, D. Mandapati, J. Davila, M. A. Coady and A. L. M. Bothwell

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Null Activity Mutation of Phenoloxidase in Drosophila melanogaster N. Asada, N. Kawamoto and T. Hatta Molecular Information Fusion for Metabolic Networks R. Hofestadt, M. Lange and U. Scholz Intron-Size and Exon Polymorphisms in the Mouse Tissue-Nonspecific Alkaline Phosphatase Gene N. Frohlander and J. L. Milldn Lipoxygenases and Cyclooxygenases of the Testis of Rat S. Neeraja, P. Reddanna and P. R. K. Reddy Effect of Heterogeneous Sperm and Hybridization of DNA Fragment in Allogynogenetic Silver Crucian Carp D. Xia, G. Xue and L. Zhang Gene Expression During Carrot Somatic Embryogenesis N. Wu, F. Diao, M. Qi, Y. Cheng, L. Zhang, M. Huang and F. Chen

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Epigenetic Modifications in Maize Parental Inbreds and Hybrids and their Relationship to Hybrid Vigor and Stability A. S. Tsaftaris, A. N. Polidoros and E. Tani

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CIS-Elements and Transcription Factors Regulating Antioxidant Gene Expression in Response to Biotic and Abiotic Signals J. G. Scandalios and L. M. Guan

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Index

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Photo 1. A rest during a picnic on an island on the Ob Sea - a man-made sea in Siberia, Russia. (I to r): Mrs. and Erwin Goldberg, John Scandalios, Clement Markert, Mrs. and Athanasios Tsaftaris, (unidentified), Eviatar Nevo, Eobert Gracy, Michael Crawford. Prof. Ma&ert was an active participant in every single congress since the inception of the Conpess series in 1%!. (The 7 th International Congress on Isozymes, Novosibirsk, Russia, September 6-13,1992).

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Photo 2. A business dinner at Oleg Serov's residence. (1 to r): Leonid Korochkin (Congress Co-Chair), Roger Homes, Oleg Serov (Congress Co-Chair), John Scandalios and Clement Markert. (The 7th International Congress on Isozymes, Novosibirsk, Russia, September 6-13,1992).

Photo 3. Clement Markert opening the 8th International Congress on Isozymes. (The 8th International Congress on Isozymes, Brisbane, Australia, June 25-July 1, 1995).

Photo 4. Margaret and Clement Markert, between sessions at the Congress Auditorium. (The 8!h International Congress on Isozymes, Brisbane, Australia, June 25-July 1,1995).

Phot© S. Sidney Atanan and Clement Markert Sid, 1989 Chemistry Mobe! laureate for the disccwery of catalytic properties of RNA, is Clement's longtime colleague and Mend. Sid was one of the six Nobel keynote speakers at the Congress to commemorate Clement's 80th birthday. (The 9 th International Congress on Isozymes, San Antonio, USA, April 14-19,1997).

Photo 6. Clement Markert speaking at a dinner to celebrate his 80 birthday. (The 9th International Congress on Isozymes, San Antonio, USA, April 14-19,1997).

Photo 7. Guoxiong Xue and Clement Markert. Guoxiong is Executive Co-Chair of the 10* International Congress on Isozymes, Genes, and Gene Families. Clement was Honorary President of the Congress. This photo was taken in 1995, Brisbane, Australia.

Photo 8. Congress banquet at a Yunan restaurant in Beijing. The Congress Chairman, Professor Zhihong Xu (President, Peking University), is taking part in one of the entertainment programs. Conspicuously absent is Prof. Clement Markert, who passed away four days before the 10* Congress commenced. (The 10* International Congress on Isozymes, Beijing, China, October 5-10,1999).

CLEMENT MARKERT 1917- 1999 GRAEME L. HAMMOND Yale University School of Medicine, Department of Surgery, 333 Cedar Street -121 FMB, New Haven, CT 06510 USA E-MAIL: [email protected] Thank you Dr. VandeBerg, members and guests of the Chinese Academy of Sciences and participants in the Congress. In April 1997, Clem Markert was diagnosed with carcinoma of the colon and underwent right hemi-colectomy. The pathology report showed 19 positive lymph nodes in a tumor that had invaded through the bowel serosa. He underwent three months of chemotherapy and was then discovered to have widespread pleural pulmonary metastases. For the next two years, he led a very active life with trips to Alaska and Africa, and boating on the Columbia and Snake Rivers. His course for the past three months, however, was one of steady deterioration - to the point that he knew he would be unable to attend the 10th International Congress. He died on the evening of October 1st, 1999 in Colorado Springs. As a surgeon and member of the Department of Surgery at Yale, I collaborated with Clem for many years during his tenure as Chairman and member of the Department of Biology at Yale. This collaboration began in an unusual way. I was investigating how the ischemic myocardium worked - an issue of great importance to medicine and to patients with coronary artery disease. During these studies, I recognized that there must be fundamental changes in the way the heart uses energy - in short, that it must be able to function anaerobically and, as the terminal step in glycolysis, be able to convert pyruvic acid to lactic acid. However, the heart never normally makes this conversion. I searched the literature for an explanation and came across papers by Clement Markert from Johns Hopkins describing the theory of isozymes and showing that the LDH isozyme pattern differed from organ to organ depending upon its energy requirements. I tried to contact Dr. Markert at Johns Hopkins and was told finally that he was, in fact, at my own institution. When I broached the idea to him that the LDH isozyme pattern in ischemic myocardium must be changing to favor lactic acid formation, he responded that this would be tantamount to Lamarckian biology. However, we later showed that the LDH pattern did change and he quickly said, "We both learned a lot from this." His openness, honesty, and high principles attracted many followers from around the world and was responsible, along with such work as his discovery of isozymes, for his election to The National Academy of Sciences.

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G. L. Hammond

Because of the unfortunate terminal nature of his disease in late September, his wife, Margaret, asked if I would give his Presidential Address which follows and which he entitled: Isozymes: A Brief Historical Perspective "Since the initial recognition of multiple molecular forms of enzymes (isozymes) by Markert and Moller (1959), isozymes have been extensively studied or used as markers in a wide range of studies, with virtually every known organism, and at all levels of biological organization. On numerous occasions since the establishment of the isozyme concept, researchers from around the world have gathered in international congresses to discuss their work and to be brought up to date on the technologies employed and the novel applications of isozymes. Each Congress has resulted in significant publications that have proved helpful to scientists from a wide range of disciplines. The study of isozymes has provided insights into the structure and function of the genome, the regulation of gene function during cell differentiation and development, and the structure, function, and evolution of isozymes and their encoding genes per se. With the advent of new technologies and developments in molecular biology came a rapid expansion in the dissection of genes encoding isozymes. Prior knowledge of isozyme structure and function served as a critical foundation of information on which research at the DNA and RNA levels could be based. The significance of gene families encoding functionally related isozymes is becoming increasingly apparent. As the genomes of various organisms from microbes to higher eukaryotes are being resolved, the question of the product of the various genes will greatly be impacted by the early and current studies with isozymes and gene families. Since the first Isozyme Congress was held in New York thirty-eight years ago (1961), there have been several revolutions in biology. Each development has had an impact on our science, and the work presented at each of the subsequent Congresses has in turn impacted biology, medicine, and agriculture in significant ways. The International Congress on Genes, Gene Families, and Isozymes has provided and will continue to provide a unique forum for international communication among biologists. As in the past, I am certain that the 10th Congress being held here in Beijing will prove to be another significant milestone in the dissemination of important information that will further enhance the use of isozymes as basic to all aspects of the biological sciences. It is obvious that isozymes will be a continuing part of biological research and will play a central role in

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Clement Markert enlarging and deepening our understanding of biological organization. A rich, rewarding, expanding, and exciting future is clearly in store for the field of isozymes as we analyze the structural and functional organization of the genome that creates the metabolic patterns which collectively make all organisms what they are." Clement L. Markert, 1999

In closing, I would like to add two personal notes. The first is from Dr. Erwin Goldberg, Professor of Biochemistry, Molecular Biology, and Cell Biology at Northwestern University, who has known Clem for many years. Dr Goldberg writes: "While discoveries in biology move the field forward, it is rare that an individual's accomplishments can have such an impact on an entire discipline. That is Clem Markert's legacy for present and future biologists." Finally, from myself, I would like to add that Clem Markert significantly affected the lives and careers of many people in this audience, including my own, and that his imprint on clinical medicine and surgery was just as great as it was on biology. His identification of isozymes is used every day in every major hospital in the world for diagnosing pulmonary emboli, myocardial infarction, and ischemia in virtually every organ. His ability to conceptualize led to our present understanding of cell and organ stress, the impending or actual presence of cell death and how to reverse these effects before death of the patient. This is Clem Markert's legacy for present and future clinicians.

IDENTIFICATION OF NOVEL GENE FAMILY MEMBERS BASED ON EFFICIENT FULL-LENGTH CDNA CLONING JIAN G U ' ' 2 , X I N - Y A N W U \ M I N Y E 2 , Q I N G - H U A Z H A N G 2 , Z E - G U A N G H A N 1 , H U A I - D O N G S O N G 3 , Y O N G - D E P E N G 1 , 3 , AND Z H U C H E N 1 ' 2

'Chinese National Human Genome Center at Shanghai, 351 Guo Shoujing Road, Pudong, Shanghai, 201203, China Shanghai Institute of Hematology, Rui-Jin Hospital, Shanghai Second Medical University, 197 Rui-Jin Road II, Shanghai, 2000250, China 3 Shanghai Institute of Endocrinology, Rui-Jin Hospital, Shanghai Second Medical University, 197 Rui-Jin Road II, Shanghai, 200025, China Email:

[email protected]

A combination of EST analysis, application of bioinformatics, primer walking, reversetranscription PCR and RACE has been widely used in obtaining novel full-length cDNAs. By applying this method we have cloned 600 novel full-length cDNAs sequences mainly from endocrine and hematopoietic systems. Some of these genes can be categorized into several gene families, which included some transcription factors and those involved in vesicle trafficking and signal transduction. There are also many novel genes showing homology to genes discovered in relatively lower creatures. The bioinformatic analysis combined with experimental methods were used for identifying new members of known gene superfamilies.

1

Introduction

Human genome project now is at a historic turning point, from structural genomics to functional genomics. According to announcement from both public sector and private company sequencing efforts, a working draft of the human genome sequence will be obtained soon, though the finishing will take some longer time [1,2]. The gene discovery and understanding of genetic information will require annotation of the sequence data using bioinformatic tools [3]. On the other hand, cloning of fulllength cDNA has been listed as one of the major tasks of the next phase of genomic science [1]. The integration of cDNA sequences into the genomic ones will greatly facilitate the identification of transcriptional units, the gene isoforms, and the mRNA level and specificity in cell/tissues as a result of genome expression. On the other hand, cDNA project links directly to the protein structural biology and exert significant impact to the medical genetics and biotechnology/pharmaceutical industries. Several approaches have been used to identify full-length cDNA, but the most efficient and popular way is EST sequencing. The conception of EST project is first proposed in the early 1980s, when some scientists recognized that short stretches of cDNA sequences could be used as marker for genes [4]. Until ten years later did this conception turn into reality with large flow of EST data output as the sequencing technique became more automatic and efficient [5]. The dbEST database bulked up 5

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in the last decade when EST projects from different tissue and diverse collection of organisms have been conducted. Along with the finishing of genomic sequencing and gene identification of some model organisms, rapid cloning of important human genes by leaping over taxonomic boundaries, from genes identified in model organisms to those embedded in the more complex genomes of human and mouse is now a possibility. Moreover, ESTs based STSs have been widely used in the construction of gene-based physical map. It also offers valuable experimental evidence of transcription when compared with other computational programs used to predict exons. So that it is a powerful tool for the prediction of exon-intron organization of genes, identification of alternative splicing events and unusual genome organization cases. Gene expression profiles in specific tissue or cell type reflect the functional features of them, hereby identification of novel genes preferentially expressed in this tissue or cell type would be important to clarify the molecular basis of certain physiological process. Functional assays of those obtained novel genes could be of great value to both research and commercial domains. Over the last 3 years, we have been undertaking projects of cataloging the expressed sequence tags (ESTs) from cDNA libraries relatively enriched in full-length cDNA of CD34+ hematopoietic stem/progenitor cells (HSPCs) populations and Hypothalamus-Pituitary-Adrenal (HPA) endocrine system. This approach turned out to be very successful in terms of both gene expression profiling and the discovery of novel genes in an efficient way [6]. Based on bioinformatic analysis, important clues could be obtained with regard to the structural and functional characteristics in the context of the cell compartments of each open reading frame (ORF) in large amount of putative fulllength cDNA sequences. Gene families and groups were identified through homology search across wide range of species through evolution. Application of bioinformatic information from public database allowed to assign the chromosomal localizations for the majority of the novel genes and to obtain the genome organizations in part of the genes, and to all at last in future. Moreover, the gene expression patterns were further approached using both "electronic Northern" and cDNA array so that genes with cell/tissue specific expression could be picked up for further functional studies. 2

2.1

Materials and Methods

EST sequencing and data analysis

CD34+ cells were harvested from cord blood and bone marrow, with gradient separation and anti-CD34 McAb-conjugated MACS (Miltenyi Biotec, Germany) separation twice. RNA extraction, lambda ZAPII cDNA libraries construction,

Identification of Novel Gene Family Members

7

Bluescript phagemid templates preparation, sequencing strategy and data management were manipulated as previous reports of our group [6]. Libraries of HPA endocrine system were constructed using classical strategy according to manufacturer's protocol (Stratagene, USA). The sequencing primers were universe primers including Ml3 Reverse and/or Forward, T3 and/orT7 primers, sequencing mix was BigDye Terminator (Perkin Elmer). 5' or 3' end ESTs generated were categorized into known-gene, known-EST and novel EST groups by BLAST searching against GenBank database with Blast and Fasta programs integrated in GCG package (Madison Wisconsin) (release 108 and later due to working time). 2.2

Full-length cDNA open reading frames cloning

The unknown-gene clones were candidates for novel full-length cDNA ORF cloning. The HSPC clone inserts sequences were obtained with combination of primer extension, partial deletion and subcloning sequencing. AutoAssembler (Perkin Elmer) was applied to assemble the sequences to get the contigs, DNA Strider (Version 1.0) was employed to analyze the reading frames of the contigs. To those partial reading frames clones, 'in silico' EST assembly and rapid amplification of cDNA ends (RACE) was efficiently applied. Proper Marathon-ready cDNA libraries (Clontech, Palo Alto, CA) were selected as RACE template, and the gene specific primers (GSP) were generated from the sequences from HSPC clone. The whole open reading frames were thus obtained and confirmed by RT-PCR. 2.3

Chromosomal mapping

Electronic mapping - For novel genes, dbEST were searched to find the hit EST, then UniGene database (http://www.ncbi.nlm.nih.gov/UniGene) was applied to determine the tissue expression pattern and chromosomal mapping of these novel genes. Those cDNA matched genomic DNA sequence data can also provide mapping information. Radiation hybrid - In addition to the electronic mapping results, Stanford G3 and GeneBridge 4 Radiation Hybrid panels (Research Genetics Inc, Huntsville, AL) was applied as a complementary method to map the novel genes [7]. The results were obtained by submitting the PCR results to the Radiation Hybrid Mapping Server at Stanford Human Genome Center (http://www-shgc.stanford.edu) and Whitehead Institute / MIT center for Genome Research (http://www-genome.wi.mit.edu/cgibin/contig/rhmapper.pl). SHGC or MIT framework markers linked to the subjected genes with a LOD score >6.0 were returned from the auto-servers.

8 2.4

J. Gu et al. Preliminarily structure and function analysis with bioinformatics

Sequence Similarity Comparison - GCG package contains the release versions of EMBL and GenBank databases where the known genes and predicted ORFs were deposited. All amino acid sequences encoded by our novel genes were searched against the nucleic acid sequence sub-databases of some important model organisms such as E.coli, S.cerevisiae, C.elegans, Drosophila, Arabidopsis, and mammals (excluding primates) with the tfasta program in GCG package, respectively. There were two reasons to choose this strategy for homology search: first, there were much more nucleic acid sequences than amino acid ones in the databases; second, through evolution, the amino acid sequences are more conservative than those of nucleic acid ones. In this study, two amino acid sequences were considered as homologues when they shared more than 25% similarity over a region of 50-100 amino acids or the Z-score value higher than 200. Fundamental Structural and Functional Elements Searching - Programs including motifs, profile scan in GCG package and prosite at the Expacy website (http://www.expacy.ch/tools/scnpsite.html) were employed to scan for the motifs on primary structure of the peptides. Programs like peptide structure, plotstructure, pepplot, coilscan and hthscan in the GCG package were applied to analyze the secondary structure of the proteins, and spscan (GCG package), signalP (http://www.cbs.dtu.dk/services/SignalP/) as well as TMHMM(http://www.cbs.dtu.dk/services/TMHMM-l-0/) were used to predict the signal peptide and the a-helix transmembrane domains in those novel ORFs so as to characterize the secreted or membrane anchored proteins. In order to acquire more information about some genes, the psort (http://www.psort.nibb.ac.jp.8800) and NNPSL (http://www.predict.sanger.ac.uk/nnpsl_mult.cgi) were chosen to predict their subcellular localization. Gene Expression Pattern - Unigene and dbEST databases were used to search for the gene expression patterns, namely as Electronic Northern. Gene expression patterns of part of these novel genes were also performed by applying Northern blotting and semi-quantitative RT-PCR. Functional assays of zinc finger genes Functional Analysis of Putative Transregulatory Domain of Construction Expression Plasmid - In order to define the transregulatory properties of zinc finger genes, we select three of them, namely ZNF191, ZNF253 and ZNF255, for further functional assay. Non-zinc finger regions of these genes were inserted into yeast plasmid pGBT9 and mammalian cell plasmid pM (Clontech). Both pGBT9 and pM contain DNA-binding domain (GAL4-BD)(l-147aa) of GAL4, which was driven by ADH1 and SV40 promoters respectively. pGBT9 or pM vector inserted with target sequences were constructed to generate fusion genes encoding GAL4-ZNF191,

Identification of Novel Gene Family Members

9

GAL4-ZNF253 and GAL4-ZNF255 chimeras, respectively. The amplified regions and the junctions in these constructs were verified by DNA sequencing. Yeast One-Hybrid System - Yeast one-hybrid system was used to detect DNAprotein interaction. Yeast reporter strain Y187 (CLONTECH), which contains an integrated lacZ reporter construct protein, was transformed with hybrid expression plasmids containing different GAL4 fusion protein, the negative control pGBT9, weak positive control pGBT9-HA (hemagglutinin) and strong positive control pCLl encoding the full-length wild-type GAL4 according to the protocol of TransActTM Assay Kit (Clontech). Qualitative and quantitative analyses of p-galactosidase were performed with the colony-lift filter assay and liquid culture assay using onitrophenyl (3-D-galactopyranoside (Sigma) as substrate, respectively. Mammalian Cell Transfection-In the mammalian assay system, the recombinant pM with different GAL4 fusions, the negative control pM and the positive control pM3-VP16 encoding herpes virus protein were cotransfected by lipofectAMIN (Gibco/BRL) into NIH3T3 or CHO cells with reporter plasmid pGAL45tkLUC containing five consensus GAL4 binding sites and thymidine kinase (TK) minimal promoter upstream of the luciferase. Different ratios of plasmids to be tested and reporter plasmid were compared in transfection assays. Analyses of luciferase were performed according to the protocol of Luciferase Assay System (Promega) and relative light unit (RLU) was measured on luminometer (Lumat LB9507). 3

Results

Totally, 50000 ESTs were generated from both CD34+ cells and endocrine system, from which 750 novel open reading frames (ORF) were obtained, which included 600 full ORFs and 150 partial ORFs. (Available on website http://www.chgc.sh.cn) Only full ORFs were submitted for further functional analysis. After homology and motifs searching, the 600 ORFs were divided into 7 functional categories according to the functions of their homologue genes or possible functional domains they contain as shown in table 1. Regarding those genes with unknown functions, we compared them to genes discovered in relatively lower model organisms from virus to plants and 151 of them showed homology as shown in table 2. While considering the average length of either full-length cDNA or their deduced peptides, we found that most of them were around 500-1500 in nucleotides and 100-300 amino acids respectively, suggesting a more efficient full-length cloning strategy should be developed to obtain longer genes.

J. Gu et al. Table 1. Functional category of genes. categories Gene number Cell division 26 Cell signaling 50 Cell structure/mobility 13 Cell/Organism defense 9 Gene/Protein expression 99 Metabolism 69 unclassified 334 Table 2. Genes with Homology to those from Lower Creatures. Creature Gene number cowpox virus 3 Bacillus subtilis 3 Haemophilus somnus 1 Saccharomyces cerevisiae 41 Caenorhabditis elegans 79 Drosophila melanogaster 13 Arabidopsis thaliana 11

Further analysis of those genes with homology to known genes reveals that part of them belong to several gene families. The biggest gene family is zinc finger and leucine zipper family, with 17 members respectively. Vesicle transporting related gene families are also abundant in our catalogues, which included 6 ras-related protein, 3 VAMP proteins, 2 sec22 protein and so on. We also identified some gene families involved in signal transduction, for example, the PKA and PTP family with 6 and 1 members respectively. Zinc finger gene family belongs to one of the largest human gene families and plays an important role in the regulation of transcription [8]. This large family may be divided into many subfamilies such as Cys2/His2 type, glucocorticoid receptor, ring finger, GATA-1 type, GAL4 type and LIM family [9-10,13]. In Cys2/His2 type zinc finger genes, there are highly conserved consensus sequence TGEKPYX (X representing any amino acid) between both zinc finger motifs. The zinc finger proteins containing this specific structure are named after kriippel-like zinc finger proteins because the structure was firstly found in the Drosophila kriippel-protein [11]. In our study, we found 14 typical C2H2 zinc finger genes and 3 ring finger ones. Bioinformatics analyses revealed that ZNF191, ZNF253, ZNF254, ZNF255, ZNF256, and ZNF257 were novel genes belonging to Kriippel-like zinc finger gene family. The deduced amino acid sequences of these genes contain 3-18 tandemly repeated zinc finger motifs related to Drosophila Kriippel gene family at the Cterminal and possible transcriptional regulatory elements such as KRAB and SCAN box at their N-terminal. The amino acid "knuckle" between zinc finger motifs, typified by the amino acid sequence TGE(R/K) P (F/Y) X, was also highly conserved in all six deduced amino acid sequences. From these features it was

11

Identification of Novel Gene Family Members

reasonable to predict that all six genes could encode DNA-binding proteins with transcriptional regulatory properties. A Novel Trans-regulatory Domain KRNB Analysis of Non ZF Regions Deduced 368 amino acid sequence of ZNF191 had 4 continuous typical krilppel-like zinc finger motifs in C-terminal and contains rich acidic amino acids in non-zinc finger region. An 81 amino acid stretch at the N-terminal of these genes was highly conserved and has been designated as the SCAN box [12]. In addition to 3, 14, 13 and 4 tandemly arranged typical Cys2Mis2 zinc finger motifs respectively, ZNF253, ZNF254, 3^F256f and 7NF257 genes contained Krtippel-associated box (KRAB) in their non-zinc finger regions. These domains consisting of approximately 75 amino acids are all located at the N-terminal moiety of the genes and enriched in hydrophobic and negatively charged residues with the L (X6)L at its core. This core isflankedby certain residues "(e.g. E, L, V, and C) that arefrequentlyfound in ahelices. Although ZNF2S5 has 18 continuously tandem zinc finger motifs homologous with KrOppel-like zinc finger, its deduced amino acid sequence contains a previously undefined domain, which consists of approximately 81 amino acids, at the N-terminal of the protein. This region was homologous with a few zinc finger genes such as FDZF2 (GenBank accession number U95044) and Q14588, which are enriched in hydrophobic amino acids (e.g. G, I, A, L, F) and negatively charged acidic amino acids (e.g. D) (Fig. 1). This new domain was thus nominated as Krappel-related novel box (KRNB). *

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figure 1. Amino acid alignment of non-zincfingerregionfromZNF255 and related proteins. Conserved amino acid residues are in same color.

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Expression Pattern of ZNF191, ZNF253, ZNF254, ZNF255, ZNF256, and ZNF257- Northern blots and semi-quantative RT-PCR were carried out to examine the tissue or cell expression pattern of these zinc finger genes (data not shown). ZNF191 gene was expressed in almost all tissues and cell lines except for heart. The other five genes were selectively expressed in different tissue. Within the hematopoietic system, ZNF191 and ZNF255 were expressed in all lineages, whereas ZNF253 expression was restricted to monocyte (U937) and immature erythroid (K562). ZNF256 and ZNF257 tended to be expressed in myelomonocytic lineages (HL-60 and U937), although a low expression level could be detected in Tlymphocytes (MOLT-4) and early erythroid cells (K562). ZNF253 expression was observed in all lineages except for K562 cells. Functional Analysis of KRNB in Comparison with SCAN and KRAB Transcriptional Regulatory Domain To further address the function of the six genes isolated in the present work, ZNF191, ZNF253 and ZNF255 were chosen to study their transcriptional regulatory activities, since these genes contain SCAN, KRAB and KRNB, respectively. The recombinant pGBT9-ZNF191 containing GAL4ZNF191 chimera and control plasmids pGBT9, pGBT9-HA, and pCLl were then used to transform Yeast strain Y187. The qualitative and quantitative analyses of Pgalactosidase indicated that ZNF-191 might be a transactivator in Y187, since a substantial activity of P-galactosidase from the GAL4-ZNF-191 chimera was observed, as compared to the controls (Fig. 2A). However, when a recombinant pM containing GAL4-ZNF191 chimera was cotransfected with a luciferase reporter plasmid into mammalian cells CHO and NIH3T3, it failed to stimulate the expression of the reporter gene. The luciferase activity was even lower than that of pM with basal activity (Fig. 2B and C). Analysis using yeast one-hybrid system and mammalian cell transfection for defining the functions of KRAB domain from ZNF253 generated, nevertheless, coherent results. After Y187 was transformed with pGBT9-ZNF253 containing GAL4 BD-ZNF253 (l-174aa) chimera, both qualitative and quantitative assays of P-galactosidase displayed a suppressive effect of ZNF253 non-zinc finger region on the transcription of reporter gene lacZ, making the galactosidase activity lower than that from pGBT9 with minimal basal stimulation. A similar transcriptional repressor effect was also observed in mammalian cells in that recombinant pM containing GAL4-ZNF253 fusion gene inhibited significantly the expression of reporter plasmid pGAL45tkLUC in CHO and NIH3T3 cell lines (Fig. 2A, B and C).

Identification of Novel Gene

Figure 2. Functional analysis of putative transregulatory domain of ZNF191, ZNF253, ZNF255 by yeast one-hybrid system and mammalian cell transfection. Each value represents the mean of three replicate assays. The error bars indicate standard deviation from the mean. Where the error bars are not visible, the standard deviation was smaller. A, quantitative analysis of |3-glactosidase in yeast reporter strain Y187 transformed with hybrid expression plasmids containing different GALA fusion protein such as GAL4 BD-ZNF191 (1-25laa), GAL4 BD-ZNF253 (l-174aa) and GAL4 BD-ZNF255 (l-81aa). Y187 were also transformed with the negative control pGBT9, weak positive control pGBT9-HA (hemagglutinin) and strong positive control pCLl encoding the full-length wild-type GAM simultaneously. B and C, analysis of luciferase in CHO and NH3T3 cells cotransfected by constructive plasmids derived from pM containing GAL4 BD with reporter plasmid pGAL45tkLUC, respectively. The recombinant pM including GAL4 BD-ZNF191 (l-251aa), GAL4 BD-ZNF253 (l-174aa) and GAL4 BD-ZNF255 (l-81aa), the negative control pM and the positive control pM3-VP16 encoding herpes virus protein were cotransfected into CHO and NM3T3 cells with different molar ratios of plasmids to be tested and reporter plasmid. Open columns and filled columns represent ratio of 1:1 and ratio of 1:3, respectively.

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To approach the property in transcriptional regulation of ZNF255 containing KRNB domain, the same experimental procedures were performed. Non-zinc finger region (l-81aa) including the KRNB domain was subcloned into the pGBT9 and pM to form in frame fusions, which were used to transform Y187 and transfect mammalian cell lines, respectively. It is interesting to note that the fusion protein GAL4-ZNF255 can stimulate the expression of reporter gene lacZ in yeast. However, slight transcriptional suppression was observed in both CHO and NIH3T3 cell lines (Fig.2A, B and C). Chromosome Localization of ZNF191, ZNF253, ZNF254, ZNF255, ZNF256, and ZNF257. Using FISH, ZNF191 was mapped on chromosome 18ql2.1. Interestingly, ZNF253, ZNF254, ZNF255, ZNF256, ZNF257, ADCAHAOl and CBCBHDIO were all mapped on chromosome 19, ZNF253, ZNF254, ZNF257 being located at 19pl3 and ZNF255, ZNF256 ,ADCAHA01 and CBCBHDIO at 19ql3 by RH technique (Fig 3).

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Figure 3. 7 novel zinc finger genes were localized on chromosome 19 by using RH and STS searching.

Identification of Novel Gene Family Members

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Ring Finger is another subfamily of zinc finger family which contains two subtypes of C3HC4 and C3H2C3 ring finger [13]. This subfamily contains several functional important tumor related genes such as PML and BRCA1 [14,15]. However, we have identified three members in our library. Further functional analysis of these genes are now undertaking. Leucine zipper is a kind of transcription factor with a characteristic L-X(6)L pattern [16]. 17 novel full-length cDNA were found to have this pattern while 6 of them have this pattern localized at a-helix or coil region. When NLS searching was performed, only 3 of them showed this signal. 4

Discussion

Since tissue- or development stage-related differential expression exists for many genes, cloning of full-length cDNA based on EST analysis in different tissues represents a useful approach for gene identification, especially for those subject to temporal-spatial regulation. In strict sense, a full-length cDNA should cover both the ORF and the complete 5' and 3' UTR. Though a number of methods have been used to surmount the technical obstacles for getting the 5' end of cDNA [17], it is still difficult to reach the transcription start site in many cases. However, as the most important functional information of the mRNA is contained in the ORF, cDNAs containing entire ORFs are often considered as being full-length. By combining several technologies including construction of full-length cDNA enriched libraries, in silico cloning and RACE, a relatively efficient working system has been established to obtain full-length cDNAs, or more precisely cDNAs including entire ORFs, in a cost-effective way. This system has enabled the first resource of cDNAs with entire ORFs to be generated for novel genes whose expression is found in human CD34+ HSPCs and neuro-endocrine system. One strong challenge to the genomic science nowadays is to elucidate the function of the newly discovered huge amount of genes. In this work, we tried to apply the currently available bioinformatic tools to the analysis of the structural and functional characteristics of each ORF. Some experimental assays were also performed to explore the functions of some important genes. Using BLAST search, totally 266 out of 600 ORF were found to share homology to genes with known functions, offering thus important clues for the choice of appropriate functional assays in further study. Hereby we divided them into several gene families involved in transcriptional regulation, vesicle transporting, signal transduction and so on. Cys2/His2 type zinc finger gene family is one of the largest gene families and each member has repeated zinc finger motifs containing finger-like structure by 2 cysteine and 2 histidine covalently binding to one zinc ion[18]. It is estimated that in this huge family, about one third of the members are Kriippel-like genes as characterized

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by the presence of highly conserved connecting sequences "TGEKPYX" between adjacent zinc finger motifs. Substantial evidence indicates that Kriippel proteins are important players in many physiological processes as transcriptional regulators. Kriippel-like zinc finger family has many subfamilies based on non-zinc finger regions and these subfamilies play distinct roles in terms of transcriptional regulation of target genes. So far several domains are found in non-zinc finger regions, such as KRAB, FAX (finger-associated box), POZ (poxvirus and zinc finger), SCAN, FAR (finger-associated repeats) and PR domains [12,19-22]. These domains may affect transcription directly or indirectly. There is evidence to show that KRAB domain, present in one-third of the Kriippel-like zinc finger genes, functions as transcriptional repressor [23]. Of note, 4 genes cloned in the present work contain KRAB domain and the experiments on ZNF253 containing KRAB domain did show transcriptional repressive activities. In view of its wide existence, it is reasonable to suggest that the KRAB domain play an important role in transcription regulation. However, results on the transregulatory properties of other domains from different authors could be controversial. In this study, the SCAN domain from ZNF191 showed distinct activities in different experimental systems, slight transactivator in yeast cells but transrepressor in CHO and NIH3T3 cells. It is possible that the properties of SCAN are determined by gene and/or cell context with distinct transcriptional machineries. A previously undefined domain, nominated here as KRNB, was discovered in ZNF255. This domain, when fused with GAM BD, upregulated the transcriptional expression of luciferase reporter gene in yeast, although no obvious effect was observed in both CHO and NIH3T3 cells. It is thus possible that the KRNB functions as a conditional transactivator. Previous work showed that in human being, more than 40 zinc finger genes aggregated on chromosome 19pl3 and more than 10 genes on chromosome 19ql3 [24-25]. Chromosomal localization also supports this conclusion because 7 of them are aggregated on chromosome 19pl3 or 19ql3 regions except for ZNF191 that has been mapped to chromosome 18. The precise functions of these genes should be further elucidated. However, exploring the function of these novel genes with homology to genes of known functions may provide an insight into their novel functions as well as confirming their known functions. One imporant clue to the possible functions of these novel genes was their expression pattern (available on website: www.chgc.sh.cn) . It is interesting to note that that ZNF253, ZNF254, ZNF256, and ZNF257 are selectively expressed in certain leukemia cell lines representing different lineages, and thus could be related to the differentiation and maturation of hematopoiesis. In contrast, ZNF191 and ZNF255 show ubiquitously expression in leukemia cell lines Regarding these novel genes without ascertained functions, bioinformatic tools are used to search the functional motifs and domains as well as their possible subcellular localization, thus speculate the possible pathways it may involve in.

Identification of Novel Gene Family Members

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The difficulty was how to deal with the majority of the ORFs without obvious functional information. We therefore attempted to evaluate the conservatism of the sequences through evolution. As a result, 151 ORF show over 25% similarity at amino acid level to those identified in organisms including E.coli, S.cerevisiae, C.elegans, Drosophila, Arabidopsis and mammals. Though a large proportion of these evolutionarily conserved genes are of unknown function, this analysis can provide at least the following information: on one hand, they are most likely to exert important biological function; and on the other hand, the low organisms containing homologous sequences can be used as models in the functional study with gene knock-out or other methods. Moreover, efforts have been made to approach the gene function by search of distinct motifs, including those related to the subcellular localizations. Regarding those orphan genes with no homologous genes available, de novo functional analysis should be taken while keeping comparison to genetic information of any newly sequenced genomes of model organisms. New approach such as more efficient functional analysis assays and 3D modeling software needs to be developed, in order to speed up the shift from structural genomics research to functional genomics research. 5

Acknowledgement

This work was supported in part by the Chinese High Tech Program (863), the Chinese National Key Program for Basic Research (973), the National Natural Science Foundation of China, Shanghai Commission for Science and Technology, and the Clyde Wu Foundation of SIH. The authors thank all members of SIH, SIE and CHGC for their constructive discussion and encouragement. Reference 1.

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Identification of Novel Gene Family Members

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18. Jacobs G.H., Determination of the base recognition positions of zinc fingers from sequence analysis. EMBO J. 11 (1992) pp.4507-4517. 19. Knochel W., Poting A., Koster M., Elbaradi T., Nietfeld W., Bouwmeester T., Pieler T., Evolutionary conserved modules associated with zinc fingers in Xenopus laevis. Proc. Natl. Acad. Sci. USA 86 (1989) pp. 6097-6100. 20. Bellefroid E.J., Poncelet D.A., Lecoq P.J., Revelant O., Martial J.A., The evolutionarily conserved Kruppel-associated box domain defines a subfamily of eukaryotic multifingered proteins. Proc. Natl. Acad. Sci. USA 88 (1991) pp.3608-3612. 21. Albagli O., Dhordain P., Deweindt C., Lococq G., Leprince D., The BTB/POZ domain: a new protein-protein interaction motif common to DNA- and actinbinding proteins. Cell Growth Differ. 6 (1995) pp.1193-1198. 22. Liu L., Shao G., Steele-Perkins G., Huang S., The retinoblastoma interacting zinc finger gene RIZ produces a PR domain-lacking product through an internal promoter. J. Biol. Chem 272 (1997) pp. 2984-2991. 23. Friedman J.R., Fredericks W.J., Jensen P.E., Speicher D.W., Huang X.P., Neilson E.G., Rauscher F.J. 3rd., KAP-1, a novel corepressor for the highly conserved KRAB repression domain. Genes Dev. 10 (1996) pp.2067-2078. 24. Shannon M., Ashworth L.K., Mucenski M., Lamerdin J.E., Branscomb S., Comparative analysis of a conserved zinc finger gene cluster on human chromosome 19q and mouse chromosome 7. Genomics 33 (1996) pp.112-120. 25. Bellefroid E.J., Lecocq P.J., Benhida A., Porcelet D.A., Belayew A., Martial J.A., The human genome contains hundreds of genes coding for finger proteins of the Kruppel type. DNA 8 (1989) pp.377-387.

STRATEGIES FOR TESTIS SPECIFIC GENE EXPRESSION ERWINN GOLDBERG Department of Biochemistry,

Molecular Biology and Cell Biology, Northwestern Evanston, 1L 60208 U.S.A. E-mail:

University,

[email protected]

The mammalian testis is a unique organ programmed for both endocrine and germ cell production, functions that are clearly interdependent. The germ cell component displays distinctive developmental properties illustrated by programmed molecular events that occur with the onset and during spermatogenesis and include activation and inactivation of numerous genes yielding protein products with distinct or modified properties. Genes expressed during spermatogenesis can be classified as "housekeeping" or structural. Both categories include testis specific isozymes and isoforms. One such example is LDHGt, a member of the lactate dehydrogenase gene family that is transcribed only during prophase of the first meiotic division. We have cloned and sequenced the promoter of this gene and demonstrated functionality. Even though this gene and protein are well-studied, there remains the question of why LDH-C4 supplants the other lactate dehydrogenases in testis and sperm metabolism. A second example of an unique protein is provided by calpastatin. This protein is the endogenous inhibitor of calpain, a cytoplasmic cysteine protease. The calpastatin gene, unlike ldhc, is the product of alternative promoter usage by which a truncated testis specific isoform of the somatic calpastatin is produced. Testis calpastatin (tCAST) is transcribed and translated in round spermatids. The promoter region and coding exon is located within an intron of the somatic gene. We have co-localized the testis calpastatin and calpain to the region of the sperm between the plasma membrane and outer acrosomal membrane where presumably it may be a player in the events associated with the acrosome reaction and/or with sperm-egg fusion. A third example is UDP-N-acetylhexosamine pyrophosphorylase, described originally as AgX, the product of an alternatively spliced mRNA. A 16 amino acid deletion in the protein product results in a change in substrate specificity. The large number of testis specific and testis abundant isozyme and protein isoforms suggests that they are not a biological curiosity, but rather are required for both full and complete spermatogenesis and for sperm function. Mechanisms regulating testis-specific gene expression, and structure/function aspects of testis gene expression will be addressed in this report.

1

Specific Gene Expression During Spermatogenesis

Development of an undifferentiated stem cell to the highly specialized spermatozoan is a complex process. Spermatogenesis occurs in three stages. First a stem cell, the spermatogonium undergoes a series of mitotic divisions resulting in renewal of the stem cell population, apoptosis, or commitment to enter the meiotic pathway. This in turn leads to the spermatocyte which in the meiotic phase undergoes two divisions that yield the haploid spermatid. The spermatid then undergoes extensive

21

22

E. Goldberg

remodeling involving cytoplasmic reduction and nuclear chromatin condensation for delivery to the egg during fertilization. In addition to well-studied morphological changes spermatogenesis is characterized by activation and repression of many genes including those encoding isozymes and protein isoforms unique to the testis [1,2]. This paper describes three examples of regulatory strategies resulting in expression of testis specific proteins. 2

Alternative Splice Variants

There are a number of examples of alternative splice variants in germ cells and other tissues 1. AgX subsequently named SPAG2 for Sperm Antigen 2, was discovered in a screen of a human testis cDNA expression library with a pool of sera containing antibodies that agglutinated spermatozoa [3]. AgX cDNAs isolated from testis and placenta cDNA libraries (AgX-1 and AgX-2, respectively) differed by a 48-bp deletion in the open-reading frame (ORF). The AgX-1 and AgX-2 ORF's encoded putative peptide chains of 505 and 521 amino acids (-55.5 and -67.3 kDa), respectively. Both AgX isoforms occur in the testis, but AgX-1 appears to be the only species present in spermatozoa. Immunofluorescence analysis of human spermatozoa detected AgX in the principal piece of the tail. Subsequently, we showed by immunoelectronmicroscopy, localization to the outer dense fibers, structural filaments associated with the mammalian sperm axoneme [4]. Southern analysis of human genomic DNA with a probe common to both AgX isoforms indicated a single AgX gene, therefore alternative splicing is the likely mechanism for production of these variant mRNAs. The AgX isoforms differed by a 16 amino acid deletion suggesting that the AgX-1 mRNA resulted from splicing out of a "miniexon", as has been suggested for mRNAs that differ by a small insertion, e.g. the CI and C2 heterogeneous ribonuclear proteins [5]. Furthermore, alternative splicing of short exons has been proposed as an "on/off switch" for the testis isoforms of other proteins such as the cyclic adenosine 3',5'-monophosphate response element binding protein (CREB) [6]. The tight control of gene expression by alternative splicing occurs frequently in differentiating tissues such as the testis [7]. When we originally described AgX3, the cDNA nucleotide and derived amino acid sequences were not similar to any sequences in the Genbank, EMBL SwissProtein, or PIR data libraries. Recently Mio et al. [8] reported that the cDNA for human UDP-N-acetylglucosamine pyrophosphorylase (UAP1) was identical to AgX. The substrate for this enzyme, N-acetylglucosamine-1-phosphate (GlcNAc-1-P), is a ubiquitous and essential metabolite and plays important roles in several metabolic processes. Subsequently, Wang-Gillam et al. [9] found that substrate specificity of these isoforms differ. The amino acid deletion in AgX-1 changes UDP-N-

Testis Specific Gene Expression

23

acetylhexosamine pyrophosphorylase specificity from UDP-GlcNAc to UDPGalNAc. The significance of this shift in substrate specificity is not immediately apparent but identification of AgX-1 as UDP-GalNAcPP should help unravel its function in spermatozoa. 3

Alternative Promoter Usage: Testis Specific Calpastatin (tCAST)

A testis specific isoform of calpastatin was identified in a screen of a human testis cDNA expression library with serum from an infertile woman [10]. Three transcripts were detected in human testes by Northern blots; the smallest of which (1.9 kb) was specific to testis [11]. The testis specific transcript contained 186 bp of unique sequence at the 5' end. The remainder of the molecule was virtually identical to somatic calpastatin. Calpastatin is the endogenous inhibitor of calpain, a widely distributed cysteine protease. The calpain/calpastatin system is Ca+2 activated and plays important roles in membrane fusion events and vesicle formation. Calpain has been detected in porcine and human sperm [12,13], suggesting critical involvement in sperm function and fertilization. The importance of Ca+2 to calpain activation and the absolute requirement for Ca+2 influx in initiating the acrosome reaction [14,15] support this contention. Isoforms of calpastatin have been described in several tissues. Differences arise from alternate splicing and exon skipping. The testis isoform of calpastatin, however, is the product of unique promoter usage and a single exon residing within intron 14 of the somatic calpastatin gene 11. The overall structure of tCAST is similar to that of the testis specific isozyme, angiotensin-converting enzyme (ACE). A testis specific mRNA encodes t-ACE [16,17] which arises from a unique promoter and single exon in intron 12 of the somatic ACE gene [18,19]. A similar transcriptional strategy generates calspermin from the gene encoding a Ca+2 /calmodulin-dependent protein kinase IV. The calspermin transcript is produced by utilization of a testis-specific promoter located within an intron of the calmodulin kinase IV gene [20]. Functional data for the testis isoforms including tCAST have yet to establish a specific role for each during spermatogenesis. In the case of tCAST, we have learned that this isoform localizes to the space between the plasma membrane and outer acrosomal membrane of the sperm [21] and appears to be associated with the acrosomal vesicle during spermiogenesis (Li & Goldberg, in preparation). As noted above, a functional role in the acrosome reaction seems plausible and is amenable to testing. The more compelling question concerns the selection of the intronic promoter that initiates t-CAST transcription. Possibly, a testis specific trans-activation factor(s) is involved in this regulatory strategy.

24 4

E. Goldberg Unique Gene Expression

The testis specific isozyme of lactate dehydrogenase (LDH-C4) has been studied extensively and has served as an important model for testis specific gene expression. Lactate dehydrogenases became the foundation for the isozyme concept which Clement Markert formulated in 1959. Since that time the LDH literature has increased logarithmically and studies on LDH have been applied to evolution, protein structure, function and diversity, clinical manifestations and gene expression. The evolution of the LDH gene family, tissue distribution of LDH isozymes and physiological implications, have been described on numerous occasions and in exquisite detail (see, for example [22]). Molecular cloning technology applied to the ldhc gene in my laboratory has confirmed the origin in mammals of ldhc as a duplication of ldha [23]. Additionally we have cloned and sequenced the promoter region of ldhc and demonstrated its function by in vitro transcription assays [24] and in vivo as a transgene construct [25]. Surprisingly, our transgene studies revealed that the promoter was active only during the pre-meiotic stages of spermatogenesis even though the protein accumulates throughout germ cell development and differentiation. Whether this is due to stable mRNA or low turnover of the protein remains to be established. Lack of a reliable germ cell culture system makes difficult analyses of testis gene expression in general and ldhc gene expression in particular. Additionally, the question of LDH-C4 function during spermatogenesis or as a sperm enzyme remains open. Our approach to this question, therefore is to target disruption of the gene by homologous recombination. Difficulties in preparing a suitable targeting construct have been resolved by sequencing the entire gene. The gene is large (14 kb) and contains an abundance of intronic repetitive elements (Olssen, unpublished observations) which tend to confound the analyses of targeting construct. Nevertheless, we (Goldberg & Millan, unpublished observations) are completing this project to obtain the ldhc-/-mutant for phenotypic analysis. 5

Summary

Specific gene expression during spermatogenesis seems to have become the norm rather than the exception. The variety of strategies reflect the complexity of the process. Alternative splice sites, alternate promoter usage and cell specific gene expression occur in many cells during development and differentiation. The uniqueness of these gene regulatory paradigms in the testis lies in timing, distribution and specialization of the final product, the spermatozoan.

Testis Specific Gene Expression 6

25

Acknowledgements

The many students who contributed to studies on LDH-C4 are acknowledged as coauthors of the publications from my laboratory. As a personal reflection, I recall that it was my first meeting with Clem Markert at an AIBS meeting in Bloomington, Indiana in 1961 that turned me on to look for multiple forms of LDH in spermatozoa. Blanco and Zinkham and I reported in Science papers simultaneously the discovery of LDH-X (its operational designation) in 1963. Subsequently, I visited with Clem at Johns Hopkins University and clarified the existence of the C subunit of lactate dehydrogenase. Clem's perception, interest and collaboration were instrumental in supporting my long association with this isozyme. This work was supported by NIH HD05863, NIH Sub-5-U54-HD29099, and P 30 HD28048. References 1. 2. 3. 4.

5.

6.

7. 8.

9.

Goldberg E., Minireview: Transcriptional regulatory strategies in male germ cells. J. Androl. 17 (1996) pp. 628-632. Hecht N.B., Molecular mechanisms of male germ cell differentiation. BioEssays 20 (1998), pp. 555-561. Diekman A.B. and Goldberg E., Characterization of a human antigen with sera from infertile patients. Biol Reprod. 50 (1994) pp. 1087-1093. Diekman A.B., Olson G., and Goldberg E., Expression of the human antigen SPAG2 in the testis and localization to the outer dense fibers in spermatozoa. Molec. Reprod. Develop. 50 (1998) pp. 284-293. Nakagawa T.Y., Swanson M.S., Wold B.J., and Dreyfuss G., Molecular cloning of cDNA for the nuclear ribonuclear particle C proteins: A conserved gene family. Proc. Natl. Acad. Sci. USA 83 (1986) pp. 2007-2011. Waeber G., Meyer T.E., LeSieur M., Hermann H.L., Gerard N., and Habener J.F., Developmental stage-specific expression of cyclic adenosine 3',5'monophosphate response element-binding protein CREB during spermatogenesis involves alternatiave exon splicing. Mol. Endocrinol. 5 (1991) pp. 1418-1430. Smith C.W.J., Patton G., and Nadal-Ginard B., Alternative splicing in the control of gene expression. Annu. Rev. Genet. 23 (1989) pp. 527-577. Mio T., Yabe T., Arisawa M., and Yamada-Okabe H., The eukaryotic UDP-NAcetylglucosamine pyrophosphorylases. /. Biol. Chem. 273 (1998) pp. 1439214397. Wang-Gillam A., Pastuszak I., and Elbein A.D., A 17-amino acid insert changes UDP-N-Acetylhexosamine pyrophosphorylase specificity from UDP-GalNAc to UDP-GlcNAc. J. Biol. Chem. 273 (1998) pp. 27055-27057.

26

E. Goldberg

10. Liang Z.G., O'Hern P.A., Yavetz B., Yavetz H., and Goldberg E., Human testis cDNAs identified by sera from infertile patients: a molecular biological approach to immunocontraceptive development. Reprod. Fertil. Develop. 6 (1994) pp. 297-305. 11. Li S., Liang Z.-G., Wang G.-Y., Yavetz B., Kim E.D., Ngai K.-L., and Goldberg E., Characterization of a membrane associated mouse testis calpastatin. Biol. Reprod. (in Press, 2000). 12. Rojas F.J., Brush M., and Moretti-Rojas I., Calpain-calpastatin: a novel, complete calcium-dependent protease system in human spermatozoa. Molec. Human Reprod. 5 (1999) pp. 520-526. 13. Schollmeyer J.E., Identification of calpain II in porcine sperm. Biol. Reprod. 34 (1986) pp. 721-731. 14. Green D.P., The induction of the acrosome reaction in guinea-pig sperm by the divalent metal cation ionophore A23187. J. Cell. Sci. 32 (1978) pp. 137-151. 15. Talbot P., Summers R.G. Hylander B.L., Keough E.M., and Franklin L.E., The role of calcium in the acrosome reaction: an analysis using ionophore. J. Exp. Zool. 198 (1976) pp. 383-392. 16. Ehlers M.R.W., Fox E.A., Strydom D.J., and Diordan J.F.,. Molecular cloning of human testicular angiotensin-converting enzyme: the testis isozyme is identical to the carboxyl-terminal half of endothelial angiotensin-converting enzyme. Proc. Natl. Acad. Sci. USA 86 (1989) pp. 7741-7745. 17. Bernstein K.E., Martin B.M., Bernstein E.A., Linton J., Striker L. and Striker G., The isolation of angiotensin-converting enzyme cDNA. J. Biol. Chem. 263 (1988) pp. 11021-11024. 18. Howard T.E., Shai S.-Y., Langford K.G., Martin B.M., and Bernstein K.E., Transcription of testicular angiotensin-converting enzyme (ACE) is initiated within the 12th intron of the somatic ACE gene. Mol. Cell. Biol. 10 (1990) pp. 4294-4302. 19. Langford K.G., Shai S.-Y., Howard T.E., Kovac M.J., Overbeek P.A., and Bernstein K.E., Transgenic mice demonstrate a testis-specific promoter for angiotensin-converting enzyme. J. Biol. Chem. 266 (1991) pp. 15559-15562. 20. Means A.R., Cruzalegui F., LeMangueresse B., Needleman D.S., Slaughter G.R., and Ono T., A Novel Ca2+/calmodulin-dependent protein kinase and a male germ cell-specific calmodulin-binding protein are derived from the same gene. Molec. Cell. Biol. 11 (1991) pp. 3960-3971. 21. Yudin A.I., Goldberg E., Robertson K.R., and Overstreet J.W., Calpain and calpastatin are located between the plasma membrane and outer acrosomal membrane of cynomolgus macaque spermatozoa. /. Androl. (in Press, 2000) 22. Markert C.L, Isozymes: model systems for analyzing the origin, evolution, regulation, and function of gene families. In: Gene Families: Structure, Function, Genetics and Evoluton. Holmes, R.S. and Lim, H.A. (Eds.) (World Scientific Publishing Co, New Jersey, 1995) pp. 3-7.

Testis Specific Gene Expression

27

23. Millan J.L., Driscoll E.E., LeVan K.M., and Goldberg E., Epitopes of human testis-specific lactate dehydrogenase deduced from a cDNA sequence. Proc. Natl. Acad. Sci. USA 84 (1987) pp. 5311-5315. 24. Zhou W., Xu J., and Goldberg E., A 60-bp core promoter sequence of murine lactate dehydrogenase C is sufficient to direct testis-specific transcription in vitro. Biol. Reprod. 51 (1994) pp. 425-432. 25. Li S., Zhou W., Doglio L., and Goldberg E, Transgenic mice demonstrate a testis-specific promoter for lactate dehydrogenase (LDH). J. Biol. Chem. 273 (1998) pp. 31191-31194.

OXIDIZED ISOFORMS AS DIAGNOSTIC BIOMARKERS OF ALZHEIMER'S DISEASE ROBERT W. G R A C Y , JOHN M. TALENT, CHRISTINA MALAKOWSKY, RACHEL D A W S O N , P A M MARSHALL, AND C R A I G C. CONRAD

Molecular Aging Unit, Department of Molecular Biology and Immunology, University of North Texas Health Science Center, Fort Worth, Texas, 76107, USA Email: [email protected] Senile plaques consist of beta-amyloid (AfJ), and is the major pathology found with Alzheimer's Disease (AD). Ap\ is particularly sensitive to oxidation, but can also produce reactive oxygen species (ROS) during Afl-fibril formation. Cells from AD subjects are more sensitive to oxidation than non-AD age-matched controls, and it appears that a number of proteins are preferentially oxidized in plasma samples from AD compared to non-AD. We are using immunoprobes specific for oxidized proteins to elucidate the mechanism of oxidative damage and apoptosis in the neuron and to evaluate the potential of oxidized isoforms as biomarkers for early detection of AD.

1

Introduction

1.1 Oxygen and ROS Oxidative metabolism is more efficient than anaerobic metabolism, however, incomplete oxygen metabolism leads to cytotoxic reactive oxygen species (ROS). There are many different types of ROS, including oxygen radicals (e.g., superoxide anion, hydroxyl radical), non-radical oxygen species (e.g., hydrogen peroxide, ozone), reactive lipids and carbohydrate derivatives (e.g., hydroxynonenal, malondialdehyde, ketoamines, or ketoaldehydes), as well as others. These ROS can spontaneously react with virtually all cellular macromolecules (e.g., proteins, lipids, and nucleic acids), causing undesirable damage and cell death. For recent reviews see: [1,2,4,5,8]. As seen in Figure 1, damaging ROS also occur from environmental exposure. For example, cigarette smoke, air/water pollutants, ozone, some food additives, and medications all contain powerful oxidizing compounds that directly cause ROS or indirectly generate ROS during breakdown and catabolism. Furthermore, low-level cosmic irradiation, x-rays and other types of electromagnetic irradiation can generate ROS. Even ultraviolet light produced by sunlight can induce photooxidations.

29

30

R. W. Gracy et al.

In vivo ROS activated astrocytes. or glial cells, etc.

EXTERNAL ROS Pollutants, UV radiation, etc.

Cell Death Figure 1. The production of Reactive Oxygen Species (ROS). Damaging oxygen/nitrogen species can be generated in vivo or from the environment. Severe damage can result in cell dealth via Necrotic, or Apoptotic pathways.

ROS are also produced by the cellular immune system to combat infections. Macrophages kill invading microorganisms by generating toxic ROS. Because ROS can damage cells indiscriminately, some of the host's cells also succumb to the macrophage attack on the invading microorganism. In the case of chronic inflammations, such as autoimmune responses, much of the tissue damage is due to ROS generated by the immune system. 1.2 ROS Damage and Aging Susceptibility to oxidative stress is more pronounced with age. Organisms accumulate oxidative damage with age, and ROS are implicated in the fundamental process of aging. This has been substantiated both in vitro and in vivo. Cells and tissues exposed to low-level ROS accumulate oxidized proteins similar to those observed in aged cells and tissues. Furthermore, when laboratory animals are fed a

Oxidized Isoforms as Diagnostic Biomarkers of Alzheimer's Disease

31

caloric-restricted diet (to increase life span), the amount of oxidative damage to cellular components is reduced [2]. This evidence supports a correlation between age and the accumulation of oxidatively damaged proteins. ROS react with DNA and cause mutations that can lead to cancer. Beckman and Ames [1] have estimated that a steady state level of DNA damage is approximately 150,000 oxidative adducts per cell, and these oxidative modifications may contribute to half of all human cancers. Furthermore, oxidized proteins may represent 30-50% of the total cellular protein of an old individual [2]. These modifications can result in peptide fragmentation, cross-linking, and amino acid modifications. Essentially every amino acid in a protein is potentially susceptible to chemical modification by oxidation. Such modifications can result in changes in the protein secondary and tertiary structures, and these conformational changes may expose previously shielded regions to further oxidations, or other types of spontaneous modifications such as deamidation [6]. The turnover of modified or damaged proteins also decreases with increasing age. Modified or damaged molecules are more readily degraded in young cells and tissues compared to similar proteins in old cells and tissues, which may interfere with the cell's ability to maintain homeostasis. The accumulation of such oxidized proteins with age was originally thought to result from random oxidation events. However, different ROS are not equally damaging to all amino acids, and different proteins exhibit different susceptibilities to such damage. Schoneich and Yang [13] have pointed out the importance of peptide sequence and neighboring groups in the oxidation potential of methioninecontaining peptides. In addition, protein oxidation can result in free radical propagation. The amyloid beta peptide (A(3) in the brains of patients with Alzheimer's Disease is an example. The A(J peptide contains 40 amino acids, of which only one methionine residue (Met35) can be "easily" oxidized [15]. In contrast, peptides that contain the same amino acids, but in the reverse sequence or scrambled sequences, do not become oxidized. This emphasizes the importance of specific amino acid sequences for susceptibility to oxidation. Because A(3 can also generate free radicals, it is believed to contribute to oxidative damage and neurotoxicicity that occur in the brains of Alzheimer's patients [7,9,10]. Substitution of cysteine for Met35 eliminates the toxic effects of Ap"s toward cells in culture [16].

32 2

R. W. Gracy et al. Results

2.1 Oxidative Damage and Alzheimer's Disease We now recognize that many of the age-related neurodegenerative diseases such as Parkinson's, Alzheimer's and other dementias are either caused, or exacerbated by oxidative damage. This can be explained because the brain is particularly susceptible to ROS damage. First, the brain relies on very large amounts of oxygen (e.g., approximately 20% of the total body oxygen consumption is for brain metabolism). Secondly, brain tissue contains a high concentration of unsaturated fatty acids that are highly susceptible to oxidation. Thirdly, the brain contains high levels of iron but has a relatively low capacity for iron binding. Iron catalyzes the spontaneous generation of ROS. Finally, the brain has relatively low levels of antioxidants. Thus, ROS may play an important role in the etiology of many types of chronic neuropathies. Figure 2 shows the pathological cascade believed to take place during the development of AD. Mutations in several different genes give rise to the abnormal production of the AP peptide, which can lead to increased ROS as discussed above [14]. For example, mutations in genes for the Amyloid Precursor Protein (APP), or in genes encoding the enzymes that cleave this protein, result in the accumulation of the Ap\ AP is believed to mediate the oxidative damage, but it is not clear whether it does this directly or indirectly (or both). Some data suggest that as the peptide undergoes aggregation to oligomers, it generates ROS as a consequence of packing of the nontoxic AP monomers into a toxic oligomer. Other studies suggest that AP causes the stimulation of glial cells (AP is not toxic to glial cells), and that the resulting hyperactivity of the glial cell generates ROS. Mutation m APR l'S-1 FS-2

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Oxidized Isoforms as Diagnostic Biomarkers of Alzheimer's Disease

33

In young cells, these processes do not result in large amounts of accumulated oxidized proteins, but in old cells, the oxidation is greater and leads to neurodegeneration. This could be due to an age-related lack of neuroprotective agent(s), the loss of antioxidants with age, or the failure of old cells to recognize and destroy oxidized proteins. Both genetic pre-disposition and environmental factors play key roles in the age of onset of AD. The addition of AB cells in culture can cause cell death (Figure 3). The cells from AD patients are more susceptible to oxidative damage than non-AD controls. 125

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At the subcellular level, damage caused by exposure to ROS and AB may lead to increased membrane permeability, which results in calcium leaking into the cell. The elevated intracellular calcium may activate calmodulin and stimulate the inducible isoform of nitric oxide synthetase. Nitric oxide in the presence of superoxide spontaneously forms peroxynitrite, which can modify tyrosine to nitrotyrosine. Such nitrotyrosine modifications could effect phosphorylation cascades, such as the hyper-phosphorylation of tau in neurofibrilary tangle formation. Ultimately, the consequences of ROS damage lead to apoptotosis of the neuron causing dementia.

34 2.2 Oxidized Isoforms as Biomarkers of Alzheimer's

R. W. Gracy et al.

Two forms of AD have been characterized; early onset (familial) and late onset (sporadic, greater than 97% of all cases). Because the pathology for both forms of AD are similar, the mechanism(s) that lead to the neuronal death due to excessive Ap* deposition are thought to be similar. The initial stages of AD begin long before clinical symptoms are apparent. The ability to detect pre-clinical AD would offer opportunities to develop and test preventative measures (e.g. antioxidants). Unfortunately, postmortem observation of brain tissue is the only reliable method to date for the 100% confirmation AD. Postmortem confirmation of AD relies on the presence of senile amyloid plaques and neurofibrillary tangles of the aggregated and phosphorylated tau protein. Clearly, predictive diagnostic biomarkers for AD are needed. Genetic biomarkers can be used to predict familial AD, but this is only a small (less that 3%) subset of patients likely to develop the disease. Furthermore, genetic biomarkers are of little use for monitoring the development, progression or prevention of AD. For such purposes, oxidized protein isoforms would offer the best potential diagnostic test. Also, the oxidative damage of AD may not be restricted to proteins in the brain since the ROS may damage cells that make up the blood brain barrier. Moreover, the antioxidant defense systems are compromised in AD brains. Thus, it is likely that specific oxidized proteins may be found in the blood or cerebral spinal fluid (CSF) of persons susceptible to or suffering from AD. The identification of such blood or CSF oxidized protein biomarkers may be the key to diagnosing AD. Furthermore, the degree of oxidation of such isoforms might be reflective of the level of progression of the disease similar to the glycosylation of hemoglobin (HbAlc) in the diabetic. We are using Western blots coupled with immunological staining to identify specifically oxidized AD protein biomarkers. We have found several potential biomarkers in the blood serum. Figure 4 shows a western blot that has been immunostained and quantified for oxidized proteins. Although the protein fingerprints are similar when stained for total protein (not shown), immunostaining reveals specific proteins were more oxidized from the AD samples compared to the age-, gender-, and race- matched controls. Figure 4A (band 1) represents one possible biomarker. The data in figure 4B show that the level of oxidation of band 1 is increased nearly 3-fold in the serum from AD patients compared to Non-AD controls. This increase in oxidation appears to be specific for the protein(s) in band 1. This specific oxidation of band 1 is apparent when band 2 is quantified. Band 2 is not specifically oxidized because there is no apparent oxidation changes when AD and non-AD controls are compared.

Oxidized Isofarms as Diagnostic Biomarkers of Alzheimer's Disease

35

2.3 Antioxidants Many antioxidants exist in vivo. These include metabolites (e.g., glutathione, NAD(P) H, cysteine), enzymes (superoxide dismutase, catalase), and vitamins (e.g., vitamin A, C, E). It has also been proposed that some proteins contain specific regions of antioxidant amino acids that serve as the last line of protection against EOS damage [11]. Since the antioxidant defenses may become compromised with age, and especially in potential AD subjects, 'antioxidants may prove to be useful in preventative therapy. For example, Vitamin E has been shown to slow the progression of the AD [12]. Estrogen replacement therapy has also been used for prevention and treatment of AD. It is now recognized that this is 'due to the antioxidant properties of estrogen. In animal models, powerful antioxidants have been reported to- reverse the damage cause by ROS. Furthermore, these compounds when administered to senile animals decreased levels of oxidized proteins in their brains, and restored short-term memory [3]. However, more research is needed before the optimal antioxidants can be prescribed. For example, it is not known which antioxidants may work best, or the optimal dosage or delivery routes.

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10

10

Figure 3. Gene expression promoted by the steroid 16,17-isoxazole derivative V in human HeLa cells transfected with ERa. Compound V (•) was compared as an estrogen with estradiol (A) and I (O). Similarly, 16-(hydroxy-methylene) estrone (X), the synthetic precursor of both I and V [1], was tested as an estrogen. Results are not shown from testing its antiestrogen activity under conditions similar to those for I (see Figure 2).

4

Discussion

When the heterocyclic pyrazole group is fused with the D-ring in estrone, it imparts strong hydrogen bonding properties to that part of the steroid molecule. This causes it to bind with increased affinity for certain steroid-binding proteins, as for example in compound I (Table I). The sterically similar isoxazole group is chemically different from pyrazole; it does not hydrogen bond. The isoxazole group in V increases the hydrophobic characteristics of the D-ring relative to estradiol. These differences can explain why compound I is a much more powerful competitive inhibitor of human placental 17 $-hydroxysteroid dehydrogenase than V, shown in earlier experiments [1]. When I was compared to V with respect to estrogen receptor binding in the present gene expression studies, the hydrophobic isoxazole ring in V appeared to cause a reversal relative to I in the binding affinities for the enzyme and estrogen receptor. Alternatively we must conclude, when D-ring analogs of estradiol interact with estrogen receptors, the effect of hydrogen bonding near the steroid D-ring does not very significantly affect receptor binding.

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Acknowledgments

We thank Mary Ann Mallon for technical assistance. This work was supported by grants from the National Institutes of Health in the United States R03 CA70515 (SA) and 5R01 DK15708 (FS), and from the Chinese Academy of Sciences (QC). This project was also supported in part by the State Basic Research Development Program (973), G200016107. References 1.

Sweet F., Boyd J, Medina O., Konderski L. and Murdock G.L., Hydrogen bonding in Steroidogenesis: Studies on new heterocyclic analogs of estrone that inhibit human estradiol 17|3-dehydrogenase. Biochem & Biophys Res Commun 180 (1991) pp. 1057-1063. 2. Murdock G.L., Warren J.C. and Sweet F., Human placental estradiol 17(Jdehydro-genase: Evidence for inverted substrate orientation ("wrong-way" binding) at the active site. Biochemistry 27 (1988) pp. 4452-4458. 3. Sweet F. and Murdock G.M., Affinity labeling of hormone-specific proteins. Endocrine Reviews 8 (1987) pp. 154-174. 4. Anstead G.M., Carlson K.E. and Katzenellenbogen J.A. The estradiol pharmaco-phore: ligand structure-estrogen receptor binding affinity relationships and a model for the receptor binding site. Steroids 62 (1997) pp. 268-303. 5. Meyer C.Y., Lutfi H.G. and Adler S., Transcriptional regulation of estrogenresponsive genes by non-steroidal estrogens: Doisynolic and allenolic acid. J Steroid Biochem 62 (1997) pp. 477-489. 6. Adler S., Waterman M.L., He X. and Rosenfeld M.G., Steroid receptormediated inhibition of rat prolactin gene expression does not require the receptor DNA-binding domain. Cell 52 (1988) pp. 685-695. 7. Ramkumar T. and Adler S., Differential positive and negative transcriptional regulation by tamoxifen. Endocrinology 136 (1995) pp. 536-542.

PROBING FOR THE BASIC OF THE LOW ACTIVITY OF THE ORIENTAL VARIANT OF LIVER MITOCHONDRIAL ALDEHYDE DEHYDROGENASE BAOXIAN W E I , AND HENRY W E I N E R

Department of Biochemistry,

1153 Biochemistry Building, Purdue University, West Lafayette, Indiana 47907-1153, USA E-mail:

[email protected]

Many Asia people possess a variant form of liver mitochondrial aldehyde dehydrogenase where a lysine replaces a glutamate at position 487 in the 500 amino acid homotetrameric enzyme. From the three-dimensional structure of the enzyme, it appeared that residue 487 interacts with two arginine residues, 264 in the same subunit and 475 in a different one. We used site directed mutagenesis to probe for why the Oriental variant had a high Km for NAD and a low specific activity. The results show that these interactions are not the sole reason for the altered properties of the Oriental variant.

1

Introduction

Abusive consumption of alcohol is a problem that exists in all populations. Though investigators have tried to explain why some individuals consume intoxicating amounts of alcoholic beverages, no definitive explanation has been presented. In contrast, it has been found that there are populations who for non-religious reasons do not consume alcohol, or if they do, it is at very low levels compared to other members of the community. It turns out that these people can not metabolize well ethanol to acetate [1,2]. The normal metabolic pathway for alcohol involves ingested ethanol being converted in the liver by the action of cytosolic alcohol dehydrogenase [3] to acetaldehyde. The acetaldehyde in turn is converted into acetate, catalyzed by liver mitochondrial aldehyde dehydrogenase (ALDH) [4]. Acetate is then utilized by liver or other tissue. Both dehydrogenases use NAD as the electron acceptor producing one mole of NADH per mole of compound oxidized. The cell must convert these NADH molecules back to NAD [5]. A person might not convert ethanol to acetate for a variety of reasons. These would include decreased activity of either dehydrogenase or impaired ability to regenerate NAD from NADH. All three of these systems have been studied and it appears that the major reason for some populations to avoid drinking alcohol containing beverages is that they are deficient in an active form of liver mitochondrial ALDH [6]. Though there are many isozymes of the enzyme in liver, it appears that the mitochondrial form is primarily responsible for acetaldehyde

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oxidation [7]. Apparently, it is the aversion to acetaldehyde that causes people to choose not to drink alcohol containing beverages. Many people from the Orient, particularly of Chinese, Japanese and Korean ancestry, "flush" after drinking ethanol. That is, their faces tend to become reddish [1, 2]. Enzyme analysis was performed on many of these individuals and they were found to be lacking the active form of liver mitochondrial ALDH. Later it was shown that these people possessed a variant of the active enzyme that differed in just one of the 500 amino acids from the active form of the enzyme. At residue 487, a lysine replaced the glutamate that is found in the active form of the 500 amino acid containing homotetrameric enzyme [6]. It was assumed that the Oriental variant of the enzyme was inactive since investigators could not detect any catalytic activity when performing gel assays normally used by investigators studying ALDH. When the tools of molecular biology became available, our laboratory cloned and expressed the active form of the human ALDH [8]. We then mutated the cDNA so it would code for the Oriental variant and expressed it in E. coli. Unexpectedly, the mutant enzyme after purification was found to possess catalytic activity. The specific activity of it was 10% of the non-Oriental form of the enzyme while the Km for NAD increased to nearly 7700 uM compare to 30 uM for the highly active enzyme [9]. Thus, under either physiological conditions or even standard assay conditions, the Oriental enzyme would essentially exhibit so little activity that it would appear to be inactive. When some Asian people who were deficient in ALDH were genotyped, it was found that they possessed genes coding for both the 487 glutamate active variant as well as the 487 lysine low-active form [10]. We later showed that these people express both forms of the enzyme indicating that the Oriental subunit was dominant over the glutamate-form [11]. Crabb's laboratory demonstrated using HeLa cells that the Oriental variant caused a decrease in activity of the glutamate containing enzyme [12]. This was the first proof that the Oriental variant was dominant in a heterotetramer. Before our knowing the structure of the enzyme, we postulated that the reason for the high Km of the Oriental enzyme could be that the positive nicotinamide ring of NAD was located near the glutamate at position 487. The binding of NAD would become difficult when the glutamate residue became a lysine. Consistent with this proposal was our finding that if we placed a neutral glutamine (Q) at this position (residue 487) a low Km, high activity mutant was produced [9]. This shows that it was not the absence of the glutamate residue (E), but the presence of the positively charged lysine (K) that caused the Oriental variant to become inactive.

Oriental Variant of Liver Mitochondrial Aldehyde Dehydrogenase

R475 in B E487 in A R264 in A

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A-siibunit

B-subunit

Figure 1. Human liver mitochondrial aldehyde dehydrogenase. Panel A shows one subunit of the homotetrameric enzyme. Residue 487 in a glutamate (E) in the active form of the enzyme'and is a lysine in the Oriental variant. Also shown are two arginine (R) residues. Panel B shows a dimer of aldehyde dehydrogenase. One of the arginines that interacts with residue 487 is found in the same subunit (R264) while the other (R475) is found in a different subunit. For purpose of illustration, one subunit is shown as a ribbon. Panel C shows the tetrameric arrangement. Two pairs of dimers make of the tetrameric enzyme.

In 1997 the three dimensional structure of the corresponding beef liver enzyme was solved by Thomas Hurley's laboratory [13], It was found, much to our surprise, that residue 487 was not located near the nicotinamide ring as we postulated. Instead, this residue was located on the surface of the subunit and formed salt bonds with two different arginine residues (R). One was located at position 264 in the same subunit and the other was at position 475 in a different subunit. This structural arrangement is presented in Figure 1 along with an illustration of just one subunit of the enzyme. The enzyme actually is a dimer of dimers. That is, two subunits interact as shown in panel B to form the tetrameric enzyme, shown in panel C. The important interactions with respect to the Oriental variant take place between subunits that form one of the dimer pairs. Since the altered properties of the Oriental variant of ALDH were not a result of the lysine at position 487 directly interfering with the binding of NAD, we undertook an investigation of the importance of the interaction between it and the

Oriental Variant of Liver Mitochondrial Aldehyde Dehydrogenase two arginine residues. Site directed mutagenesis was employed. results are presented in this chapter.

2

145 Some of the

Methods

Single mutations were created by use of oligonucleotides and polymerase chain reaction techniques. The mutant colonies were selected by double stranded DNA sequencing with a thermocycler sequencing kit. Double and triple mutants were constructed by exchanging the cDNA fragments containing the single mutants with the corresponding fragments of the native or E487K cDNA from pT7-7 plasmid. All mutants forms of the enzyme were purified as described previously[14]. The purity of the enzymes was checked by SDS-polyacrylamide gel electrophoresis using the Coomassie Blue staining procedure. The final protein concentration was determined with a Bio-Rad protein assay kit with bovine serum albumin as a standard. Dehydrogenase activity assays were performed by measuring the rate of increase in the fluorescence of NADH formation in 100 mM sodium phosphate(pH7.4) at 25 °C with an Aminco filter fluoro meter. Concentrations of NAD were l-10mM for native and different mutant enzymes. The propionaldehyde concentration was 14 uM. All kinetic measurements were performed at least three times, and the mean values were used for calculations or plots. Kinetic parameters were obtained from the MicroMath scientist program. All detailed description of the methods and materials can be found in our recent publication [15]. 3

Results and Discussion

We previously demonstrated that the recombinantly expressed version of the Oriental variant of human mitochondrial liver aldehyde dehydrogenase was active but had a very high Km for NAD. It bound NAD poorly but bound NADH well [9]. Thus it appears that the enzyme has difficulty in interacting with the positive charge of the nicotinamide ring of NAD. The properties of the enzyme as well as that of the E487Q mutant are presented in Table 1. The latter construct, mentioned above, was studied so we could determine if it was the loss of the glutamate residue or the presence of the lysine that caused the enzyme to possess altered properties. From the data, it is apparent that the presence of a positive lysine hurt the enzyme and not the loss of the negative glutamate.

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From the structure shown in Figure 1 it was apparent, so we thought, that the disruption of the interactions between the glutamate at position 487 and the arginines at positions 264 and 475 were the cause of the altered properties of the Oriental variant. To test for this, a number of mutations to the enzyme were made. These included changing the arginines so that there would not be positive charge repulsions between the groups as well as trying to restore the salt bonds by introducing a negative charge in place of the positive arginines. First, simple controls were made which included changing the arginine in the native enzyme. All the data is summarized in Table 1. The arginine at position 264 does not seem to be important for the activity of the enzyme. This is not the case with the arginine at position 475. The mere removal of this arginine caused the enzyme to have altered properties. Thus, it was not possible for us to determine how important were the interactions between residue 487 and 475. It is of possible interest to note that the glutamate at 487 and the arginine at position 475 are not conserved in all the members of the ALDH family. Every form of the enzyme that has a glutamate at 487 has an arginine at position 475. This interaction, then, is critical for the enzyme. It was our hope to be able to explain why the Oriental variant of the ALDH had a low activity and a high Km for NAD. Based on structural information it appeared that the interaction between the lysine at position 487 in the Oriental variant and the arginine 264 in the same subunit or arginine 475 in the subunit making up the dimer-pair was critical. The mutational approach did not allow us to unravel this interesting question for any change made to the arginine at position 475 caused the enzyme to have altered properties. Thus in spite of knowing the three dimensional structure of the enzyme, we still cannot explain how the one amino acid replacement caused the Oriental variant to have such different properties. Table 1 Kinetic properties of various mutant of human liver mitochondrial ALDH.

Mutant E487a (Native enzyme) E487Q E487,R475Q E487.R264Q E487,R475Q, R264Q E487,R475E E487,R264E E487,R475E, R264E E487K E487K, R475Q E487K, R264Q

KM NAD (pM) 37 90 850 60 1300 1300 740 16000 7400 1500 1400

*C