Table Of ContentPROGRESS IN BRAIN RESEARCH
VOLUME 71
NEURAL REGENERATION
EDITED BY
F. J. SEIL
Ofice of Regeneration Research Programs, Veterans Administration Medical Center, and
Departmeni of Neurology, Oregon Health Sciences University, Portland, OR 97201, USA.
E. HERBERT
Institute for Advanced Biomedical Research, Oregon Health Sciences University. Portland, OR 97201, U.S.A.
and
B. M. CARLSON
Department of Anatomy and Cell Physiology, The University of Michigan Medical School, Ann Arbor,
MI 48109, U.S.A.
ELSEVIER
AMSTERDAM - NEW YORK - OXFORD
1987
0 1987, Elsevier Science Publishers B.V. (Biomedical Division)
All rights reserved. No part of this publication may be reproduced, stored in a retrieval system or transmitted
in any form or by any means, electronic, mechanical, photocopying, recording or otherwise without the prior
written permission of the publisher, Elsevier Science Publishers B.V. (Biomedical Division), P.O. Box 1527,
loo0 BM Amsterdam, The Netherlands.
Special regulations for readers in the USA
This publication has been registered with the Copyright Clearance Center Inc. (CCC), Salem, Massachusetts.
Information can be obtained from the CCC about conditions under which photocopying of parts of this
publication may be made in the USA. All other copyright questions, including photocopying outside the
USA, should be referred to the publisher.
ISBN 0-444-80814-0 (volume)
ISBN 0-444-80104-9 (series)
Published by:
Elsevier Science Publishers B.V. (Biomedical Division)
P.O. Box 21 1
loo0 AE Amsterdam
The Netherlands
Sole distributors for the USA and Canada:
Elsevier Science Publishing Company, Inc.
52 Vanderbilt Avenue
New York, NY 10017
USA
Library of Congress Cataloging-in-Publication Data
Neural regeneration.
(Progress in brain research ; v. 71)
Based on a symposium held in Pacific Grove, Calif.
on Dec. 8-12, 1985, sponsored by the Medical Research
Service of the U.S. Veterans Administration and by the
Paralyzed Veterans of America.
Includes bibliographies and index.
1. Nervous system--Regeneration--Congresses.
2. Nerves--Growth--Congresses. 3. Neuroplasticity--
Congresses. I. Seil, Fredrick J. 11. Herbert E.
(Edward), 1926- . 111. Carlson, Bruce M.
IV. Unitedstates. Veterans Administration. Medical
Research Service. V. Paralyzed Veterans of America.
VI.Series. [DNLM: 1. Nerve Regeneration-congresses.
W1 PR667J v.71 / WL 102 N4937 19851
QP376.m vol. 71 61T.82 s [599'.0188] 86-32842
[QP363.5]
ISBN 0-444-80814-0 (US.)
Printed in The Netherlands
V
List of Contributors
A. J. Aguayo, Neurosciences Uht, McGill University and The Montreal General Hospital, 1650 Cedar Ave-
nue, Montreal, Quebec, H3G 1A4 Canada
M. J. Anderson, Department of Pharmacology and Therapeutics, The University of Calgary, 3330 Hospital
Drive N.W., Calgary, Alberta, T2N 4N1 Canada
D. T. Anthony, The Rockefeller University, 1230 York Avenue, New York, NY 10021, USA
M. D. Ard, Department of Anatomy and Neurobiology, Washington University School of Medicine, 660 S.
Euclid Avenue, St. Louis, MO 831 10, USA
K. F. Barald, Department of Anatomy and Cell Biology, Program in Neuroscience, University of Michigan
Medical School, Ann Arbor, MI 48109, USA
Y.-A. Barde, Max-Planck-Institute for Psychiatry, Department of Neurochemistry, D-8033 Planegg-Mar-
tinsried, FRG
J. J. Bernstein, Laboratory of Central Nervous System Injury and Regeneration, Veterans Administration
Medical Center and Departments of Physiology and Neurosurgery, The George Washington University
School of Medicine, Washington, DC 20422, USA
A. Bjorklund, Department of Histology, University of Lund, S-223 62 Lund, Sweden
S. S. Bock, Department of Cell Biology, Vanderbilt University Medical School, Nashville, TN 37232, USA
G. M. Bray, Neurosciences Unit, McGill University and The Montreal General Hospital, 1650 Cedar Avenue,
Montreal, Quebec, H3G 1A4 Canada
R. L. Brinster, Laboratory of Reproductive Physiology, School of Veterinary Medicine, University of Penn-
sylvania, Philadelphia, PA 19103, USA
P. Brundin, Department of Histology, University of Lund, S-223 62 Lund, Sweden
M. B. Bunge, Department of Anatomy and Neurobiology, Washington University School of Medicine, 660
S. Euclid Avenue, St. Louis, MO 831 10, USA
R. P. Bunge, Department of Anatomy and Neurobiology, Washington University School of Medicine, 660
S. Euclid Avenue, St. Louis, MO 83110, USA
P. R. Burgess, Department of Physiology, University of Utah School of Medicine, Salt Lake City, UT 84132,
USA
D. W. Burmeister, Department of Pharmacology, Columbia University College of Physicians and Surgeons,
New York, NY 10032, USA
G. Buzsaki, Department of Physiology, University Medical School, P&, Hungary
T. J. Collier, Department of Neurobiology and Anatomy, University of Rochester School of Medicine and
Dentistry, Rochester, NY 14642, USA
W. F. Collins, 111, Department of Neurobiology and Behavior, State University of New York, Stony Brook,
NY 11794, USA
M. Comb, Department of Molecular Biology, Massachusetts General Hospital, Boston, MA 021 14, USA
J. R. Connor, Laboratory of Central Nervous System Injury and Regeneration, Veterans Administration
Medical Center and Departments of Physiology and Neurosurgery, The George Washington University
School of Medicine, Washington, DC 20422, USA
B. Costello, Department of Cell Biology, Vanderbilt University Medical School, Nashville, TN 37232, USA
B. A. Cunningham, The Rockefeller University, 1230 York Avenue, New York, NY 10021, USA
A. M. Davies, St. Georges Medical School, Department of Anatomy, Cranmer Terrace, Tooting, London
SW17 ORE, England, UK
B. P. Davis, Division of Molecular Biology, Department of Biochemistry, The University of Texas Health
Science Center at Dallas, Dallas, TX 75235, USA
A. Y. Deutch, Department of Pharmacology, Yale University School of Medicine, New Haven, CT 06511,
USA
J. D. Elsworth, Department of Pharmacology, Yale University School of Medicine, New Haven, CT 06511,
USA
L. F. Eng, Department of Pathology, Veterans Administration Medical Center and Stanford University
School of Medicine, Palo Alto, CA 94304, USA
L. L. Englander, The Rockefeller University, 1230 York Avenue, New York, NY 10021, USA
VI
J. R. Fallon, Department of Neurobiology, Stanford University School of Medicine, Stanford, CA 94305,
USA
B. W. Festoff, Department of Neurology, University of Kansas Medical Center and Neurobiology Research
Laboratory, Veterans Administration Medical Center, Kansas City, MO 64128, USA
C. ffrench-Constant,M edical Research C o dD evelopmental Neurobiology Project, Zoology Department,
University College London, London WClE 6BT, England, UK
M. S. Flaster, Departments of Anatomy and Cell Biology and Psychiatry, Center for Neurobiology and
Behavior, College of Physicians and Surgeons of Columbia University and New York State Psychiatric
Institute, 722 West 168th Street, New York, NY 10032, USA
D. S. Forman, Department of Anatomy, Uniformed Services University of the Health Sciences, 4301 Jones
Bridge Road, Beth&, MD 20814-4799, USA
J. A. Freeman, Department of Cell Biology, Vanderbilt University Medical School, Nashville, TN 37232,
USA
H. Fujisawa, Department of Anatomy, Kyoto Prefectural University of Medicine, Kawaramachi Hirokoji,
Kamikyo-ku, Kyoto 602, Japan
F. H. Gage, Department of Neurosciences,S chool of Medicine, University of California, San Diego, La Jolla,
CA 92093, USA
P. J. Gebicke-Haerter, Department of Neurobiology, Stanford University School of Medicine, Stanford, CA
94305, USA
A. Gorio, Fidia Neurobiological Research Laboratories, Via Ponte della Fabbrica 3/A, 35031, Abano Terme,
Italy
B. Grafstein, Department of Physiology, Cornell University Medical College, New York, NY 10021, USA
V. Greenberger, Center for Neurosciences’and Behavioral Research, The Weizmann Institute of Science,
Rehovot, Israel
S. N. Haber, Department of Neurobiology and Anatomy, University of Rochester, School of Medicine and
Dentistry, Rochester, NY 14642, USA
R. E. Hammer, Laboratory of Reproductive Physiology, School of Veterinary Medicine, University of Penn-
sylvania, Philadelphia, PA 19103, USA
D. Hantai, I.N.S.E.R.M., U 153, Biologie et Pathologie Neuromusculaires, 17 Rue du Fer-a-Moulin, Paris
75005, France
E. Herbert, Institute for Advanced Biomedical Research, Oregon Health Sciences University, Portland, OR
97201, USA
R. M. Herndon, Center for Brain Research, University of Rochester Medical School, Rochester, NY 14642,
USA
S. Hoffman, The Rockefeller University, 1230 York Avenue, New York, NY 10021, USA
K. W. Horch, Department of Physiology, University of Utah School of Medicine, Salt Lake City, UT 84132,
USA
J. D. Houle, Department of Neurological Surgery and Neuroscience, College of Medicine, University of
Florida, Gainesville, FL 32610, USA
M. J. Ignatius, Department of Neurobiology, Stanford University School of Medicine, Stanford, CA 94305,
USA
J. C. Jay, Department of Anatomy and Cell Biology, Program in Neuroscience, University of Michigan
Medical School, Ann Arbor, MI 48109, USA
W. M. Jenkins, Coleman Laboratory, Departments of Physiology and Otolaryngology, University of Cali-
fornia at San Francisco, San Francisco, CA 94143, USA
J. E. Johnson, Max-Planck-Institute for Psychiatry, Department of Neurochemistry, D-8033 Planegg-Mar-
tinsried, FRG
M. I. Johnson, Departments of Pediatrics, Neurology, Anatomy and Neurobiology, Washington University
School of Medicine, 660 S. Euclid Avenue, St. Louis, MO 831 10, USA
M. J. Katz, Bio-architectonicsC enter, Case Western Reserve University School of Medicine, Cleveland, OH
44106, USA
N. Kleitman, Department of Anatomy and Neurobiology, Washington University School of Medicine, 660
S. Euclid Avenue, St. Louis, MO 831 10, USA
J. D. Kocsis, Department of Neurology, Yale University School of Medicine and Veterans Administration
Medical Center, West Haven, CT 06510, USA
R. L. Lappin, The Rockefeller University, 1230 York Avenue, New York, NY 10021, USA
R. J. Lasek, Bio-architectonicsC enter, Case Western Reserve University School of Medicine, Cleveland, OH
44106, USA
I. M. Lieberburg, Department of Medicine, Albert Einstein College of Medicine, Bronx, NY 10461, USA
VII
R. M. Lindsay, Sandoz Institute for Medical Research, Gower Place 5, London WClE 6BN, England, UK
D. Liston, Institute for Advanced Biomedical Research, Oregon Health Sciences University, Portland, OR
9720 1, USA
S. K. Ludwin, Department of Pathology (Neuropathology), Queen’s University and Kingston General Hos-
pital, Kingston, Ontario, K7L 3N6 Canada
R. J. MacDonald, Division of Molecular Biology, Department of Biochemistry, The University of Texas
Health Science Center at Dallas, Dallas, TX 75235, USA
C. Magill, Department of Neurobiology, Stanford University School of Medicine, Stanford, CA 94305, USA
M. Martin, Institute for Advanced Biomedical Research, Oregon Health Sciences University, Portland, OR
97201, USA
B. N. Mayes, Department of Cell Biology, Vanderbilt University Medical School, Nashville, TN 37232, USA
C. M. McGuinness, 11 Freeman Street, New Brunswick, NJ 08901, USA
C. B. McGuire, Department of Cell Biology, Vanderbilt University Medical School, Nashville, TN 37232,
USA
U. J. McMahan, Department of Neurobiology, Stanford University School of Medicine, Stanford, CA 94305,
USA
L. M. Mendell, Department of Neurobiology and Behavior, State University of New York, Stony Brook,
NY 11794, USA
M. M. Merzenich, Coleman Laboratory, Departments of Physiology and Otolaryngology, University of Cali-
fornia at San Francisco, San Francisco, CA 94143, USA
N. W. Milgram, Center for Neurosciences and Behavioral Research, The Weizmann Institute of Science,
Rehovot, Israel
I. F. Mizukami, Department of Anatomy and Cell Biology, Program in Neuroscience, University of Michigan
Medical School, Ann Arbor, MI 48109, USA
R. J. Morris, Laboratory of Neurobiology and Development, National Institute for Medical Research, Mill
Hill, London NW7 IAA, England, UK
J. B. Munson, Department of Neuroscience,U niversity of Florida College of Medicine, Gainesville, FL32610,
USA
B. A. Murray, The Rockefeller University, 1230 York Avenue, New York, NY 10021, USA
0. Nilsson, Department of Histology, University of Lund, S-223 62 Lund, Sweden
R. M. Nitkin, Department of Neurobiology, Stanford University School of Medicine, Stanford, CA 94305,
USA
J. J. Norden, Department of Cell Biology, Vanderbilt University Medical School, Nashville, TN 37232, USA
D. M. Ornitz, Howard Hughes Medical Institute Laboratory, Department of Biochemistry, University of
Washington, Seattle, WA 98195, USA
R. D. Palmiter, Howard Hughes Medical Institute Laboratory, Department of Biochemistry, University of
Washington, Seattle, WA 98195, USA
L. M. Partlow, Department of Pharmacology, Veterans Administration Medical Center and University of
Utah School of Medicine, Salt Lake City, UT 84132, USA
G. W. Perry, Department of Physiology and Biophysics, University of Miami, Miami, FL 33101, USA
G. D. Phillips, Department of Anatomy and Cell Biology, Program in Neuroscience, University of Michigan
Medical School, Ann Arbor, MI 48109, USA
R. E. Pitas, Gladstone Foundation Laboratories for Cardiovascular Research, University of California, San
Francisco, P.O. Box 40608, San Francisco, CA 94140, USA
M. C. Raff, Medical Research Council Developmental Neurobiology Project, Zoology Department, Univer-
sity College London, London WClE 6BT, England, UK
G. Raisman, Laboratory of Neurobiology and Development, National Institute for Medical Research, Mill
Hill, London NW7 IAA, England, UK
D. E. Redmond, Jr., Department of Psychiatry, Yale University School of Medicine, New Haven, CT 0651 1,
USA
P. J. Reier, Department of Neurological Surgery and Neuroscience, College of Medicine, University of Flor-
ida, Gainesville, FL 32610, USA
N. E. Reist, Department of Neurobiology, Stanford University School of Medicine, Stanford, CA 94305,
USA
E. Roberts, Department of Neurobiochemistry, Beckman Research Institute of the City of Hope, Duarte, CA
91010, USA
R. H. Roth, Department of Pharmacology, Yale University School of Medicine, New Haven, CT 0651 1, USA
L. L. Rubin, The Rockefeller University, 1230 York Avenue, New York, NY 10021, USA
S. Schacher, Departments of Anatomy and Cell Biology and Psychiatry, Center for Neurobiology and Be-
VIII
havior, College of Physicians and Surgeons of Columbia University and New York State Psychiatric In-
stitute, 722 West 168th Street, New York, NY 10032, USA
R. H. Scheller, Department of Biological Sciences, Stanford University, Stanford, CA 94305, USA
A. Seasholtz, Institute for Advanced Biomedical Research, Oregon Health Sciences University, Portland, OR
9720 1, USA
M. Segal, center for Neurosciences and Behavioral Research, The Weianann Institute of Science, Rehovot,
Israel
E. M. Shooter, Department of Neurobiology, Stanford University School of Medicine, Stanford, CA 94305,
USA
M. Shyamala, Department of Biological Sciences, Stanford University, Stanford, CA 94305, USA
J. R. Sladek, Jr., Department of Neurobiology and Anatomy, University of Rochester School of Medicine
and Dentistry, Rochester, NY 14642, USA
G. J. Snipes, Department of Cell Biology, Vanderbilt University Medical School, Nashville, TN 37232, USA
J. R. Sparrow, The Rockefeller University, 1230 York Avenue, New York, NY 10021, USA
L. J. Stensaas, Department of Physiology, University of Utah School of Medicine, Salt Lake City, UT 84132,
USA
0. Steward, Departments of Neurosurgery and Physiology, University of Virginia School of Medicine, Char-
lottesville, VA 22908, USA
L. E. Swenarchuk, Department of Pharmacology and Therapeutics, The University of Calgary, 3330 Hospital
Drive N.W., Calgary, Alberta, T2N 4N1 Canada
G. H. Swift, Division of Molecular Biology, Department of Biochemistry, The University of Texas Health
Science Center at Dallas, Dallas, TX 75235, USA
S. Temple, Medical Research Council Developmental Neurobiology Project, Zoology Department, University
College London, London WClE 6BT, England, UK
H. Thoenen, Max-Planck-Institute for Psychiatry, Department of Neurochemistry, D-8033 Planegg-Mar-
tinsried, FRG
G. Thomas, Institute for Advanced Biomedical Research, Oregon Health Sciences University, Portland, OR
97201. USA
S. Varon, Department of Biology, School of Medicine, University of California, San Diego, La Jolla, CA
92093. USA
M. Vidal-Sam, Neurosciences Unit, McGill University and The Montreal General Hospital, 1650 Cedar
Avenue, Montreal, Quebec, H3G 1A4 Canada
M. Vitadello, Fidia Neurobiological Research Laboratories, Via Ponte della Fabbrica 3/A, 35031, Abano
Terme, Italy
P. D. Wall, Cerebral Functions Research Group, Department of Anatomy, University College London,
Gower Street, London WClE 6BT, England, UK
B. G. Wallace, Department of Neurobiology, Stanford University School of Medicine, Stanford, CA 94305,
USA
S. G. Waxman, Department of Neurology, Yale University School of Medicine and Veterans Administration
Medical Center, West Haven, CT 06510, USA
L. R. Williams, Department of Biology, School of Medicine, University of California, San Diego, La Jolla,
CA 92093, USA
P. M. Wood, Department of Anatomy and Neurobiology, Washington University School of Medicine, 660
S. Euclid Avenue, St. Louis, MO 63110, USA
C.-F. Zhou, Institute of Physiology, Chinese Academy of Science, Shanghai, China
IX
Preface
Research in neural regeneration is progressing at a rapid pace, and the disciplines and
techniques that are being brought to bear on this area of investigation are continuously
expanding. One of the aims of this book is to reflect the diversity of approaches to this
complex field, as well as to present as much of the current work as possible. The volume
is divided into seven sections, in part as a guide to organization of the subject matter
and in part as a grouping of the disciplines represented.
This volume includes the invited presentations at an international symposium on
neural regeneration held at the Asilomar Conference Center, Pacific Grove, California
from December 8-12, 1985. The symposium was sponsored by the Medical Research
Service of the U.S. Veterans Administration (Dr. Richard J. Greene, Director) and by
the Paralyzed Veterans of America (Mr. R. Jack Powell, Executive Director).The joint
and cooperative sponsorship of these organizations made the symposium possible. The
Program Planning Committee consisted of members of the VA Office of Regeneration
Research Programs Advisory Board and staff, including Drs. Kevin D. Barron, Jerald
J. Bernstein, Bruce M. Carlson, Charles K. Meshul, Fredrick J. Seil, Charles R. Shear
and Stephen G. Waxman, and the National Research Director of the PVA, Ms. Lynn
Phillips. The strength and breadth of the program are largely a product of the efforts
of this group. The sections on molecular neurobiology (Dr. Edward Herbert) and basal
lamina and neuromuscular regeneration (Dr. Bruce M. Carlson) were organized by my
co-editors, and both their organizational and editorial contributions were invaluable.
Dr. W. Maxwell Cowan presented an inspiring keynote address to set the tone for the
symposium. Dr. Georg Kreutzberg, who was the scheduled summary speaker, was un-
able to attend because of an injury, and was missed.
For encouragement, I am indebted to Dr. Graham V. Lees, representing the publisher,
and for technical assistance to Dr. Charles K. Meshul and Ms. Gail Rasmussen of the
VA Office of Regeneration Research Programs staff, and Dr. Robert E. Allen of the
VA Medical Research Service Central Office staff. I am grateful to all of the contributors
who labored to turn in their manuscripts in timely fashion. The results of their efforts,
as set forth in this volume, speak for themselves. Finally, I would like to thank all of
those who attended the symposium, who participated in the discussions, and who pre-
sented posters. It is unfortunate that the length of the volume precludes publication of
the excellent posters, but their presence contributed greatly to the overall edification
and excitement of the symposium, and helped provide impetus to organize future such
meetings.
Fredrick J. Seil, M.D.
Director, VA Ofice of
Regeneration Research Programs
F. J. Seil, E. Herbert and 8. M. Carlson (Eds.)
Progress in Brain Research, Vol. 71
0 1987 Elsevier Science Publishers B.V., Biomedical Division 3
CHAPTER 1
Targeted expression of cloned genes in transgenic mice
Raymond J. MacDonald", Galvin H. Swift", Robert E. Hammerb, David M. Ornitz",
Brian P. Davis", Ralph L. Brinsterb and Richard D. Palmiter"
'Division of Molecular Biology, Department of Biochemistry, The University of Texas Health Science Center at Dallas,
Dallas, TX 75235, bL.aboratory of Reproductive Physiology, School of Veterinary Medicine. University of Pennsylvania, Philadelphia,
PA 19103 and 'Howard Hughes Medical Institute Laboratory, Department of Biochemistry, University of Washington, Seattle,
WA 98195, U.S.A.
Introduction lar weight) enzymes that are structurally and func-
tionally homologous. The genes of the pancreatic
The differential expression of genes that determine serine proteases are expressed selectively in the aci-
cellular phenotype requires molecular mechanisms nar cells of the pancreas as part of the differentia-
that define the timing, extent and cell specific tran- tive phenotype of this cell type. Moreover, expres-
scription of these genes. An understanding of tran- sion in the acinar cells of the pancreas is very high:
scriptional regulation will include an understanding about 20% of the total protein synthesis of the
of the nature of DNA control sequences associated gland is for these few serine proteases (Van Nest et
with regulated genes, the number and nature of al., 1980). With the exception of kallikrein (Ashley
trans acting factors that interact with those se- and MacDonald, 1985), the presence of similar en-
quences, the nature of that interaction, and how zyme activities in other tissues appears due to
that interaction then modulates gene transcription. expression of the protein products of related but
Our approach has been to analyze tissue specific distinct genes.
control of genes through the reintroduction of As would be expected for a tissue that synthesizes
cloned genes into animals. We have chosen to study a few prominent protein products, the polyadeny-
the expression of a family of pancreas-specific lated RNA population of the pancreas contains a
genes, the pancreatic serine proteases, because of few dominant mRNAs. Figure 1 shows the profile
the advantages that accrue from the comparative of polyadenylated RNA resolved by electrophoresis
analysis of similarly regulated genes. in an agarose gel containing the denaturant meth-
ylmercury hydroxide. Major mRNAs for the secre-
Tissue specific expression of the pancreatic serine tory enzymes lipase, amylase, three carboxypepti-
proteases dases and elastase I are identified in Fig. 1. The
mRNAs for chymotrypsins, trypsins, elastase 11,
At least nine distinct serine protease genes (three kallikrein and RNase comprise a group of promi-
chymotrypsins, three trypsins, two elastases and nent RNAs ranging in length from 900 to 1,100
one kallikrein) are expressed .in the exocrine pan- nucleotides that are not resolved by electrophoresis.
creas of the rat. The extended serine protease gene The level of elastase I mRNA in several rat tis-
family has evolved from a common ancestral serine sues illustrates the tissue-specific expression of a
protease gene through a series of duplications (Neu- representative member of the pancreatic serine pro-
rath, 1984). The pancreatic family members are a tease gene family (Table 1). In the pancreas, elastase
subfamily of simple, small (25,OOO-28,000 molecu- I mRNA comprises about 1% of the total mRNA,
4
._ TABLE 1
In
Elastase I mRNA levels in the rat
-
w
VII mRNAs per cell
.I I 1
Pancreas 10,Ooo
Liver 10
Kidney 4
Intestine 3
Spleen 0.1
Submaxillary gland 10.5
Parotid <0.1
Testes <0.1
mRNA levels were quantified by solution hybridization using a
1 single stranded rat elastase I cDNA probe. The numbers of elas-
tase I mRNA molecules per cell were calculated as described by
Swift et al. (1984b).
, J , \
LENGTH (kb)
,
2.0 1.1 1.2 1.0 0.E 0.6
0 I 1'
mally on in the pancreas, and off in tissues such as
2 4 6 8
DISTANCE (cm) the parotid and submaxillary gland.
Fig. 1. Prominent mRNAs for the secretory enzymes of the rat At least one level of regulation of the elastase I
pancreas. Total RNA was isolated from rat pancreas by the gene is transcriptional. Nuclear run-on experiments
guanidine thiocyanate procedure of Chirgwin et al. (1979). Po-
detect elastase I gene transcription in nuclei isolated
lyadenylated RNA was selected by affinity chromatography on
from pancreas but not from liver (Fig. 2). Elastase
oligo-(dT) cellulose (Aviv and Leder, 1972) and resolved by elec-
trophoresis in a 1.5% agarose gel containing the denaturant I gene transcription in isolated pancreatic nuclei
methylmercury hydroxide (Bailey and Davidson, 1976). Small accounts for about 0.2% of the total, whereas tran-
amounts of 18s and 28s ribosomal RNAs remained in this po- scription of the liver specific transcript albumin is
lyadenylated RNA preparation. The major mRNAs have been
undetectable. Conversely, elastase I gene transcrip-
identified: lipase, procarboxypeptidase A and chymotrypsin B
tion is undetectable in liver nuclei in which albumin
(Bell et al., 1984); amylase (Chirgwin et al., 1979); elastases I
and I1 (MacDonald et al., 1982b); trypsin (MacDonald, 1982a); gene transcription is prevalent. The limitations of
and ribonuclease (MacDonald et al., 198%). the in vitro run-on transcription assay prevent con-
fident measurements of transcription below 1 ppm,
so the apparent level of differential transcription is
or about 10,000 mRNAs per cell. Levels are at least only 150-fold, but is probably greater. Therefore,
1,000-fold lower in other tissues such as intestine, differential rates of transcription alone may ac-
kidney and liver. The physiological significance of count for the 1,000-fold difference in elastase I
this low, but detectable, expression in these tissues mRNA levels of the pancreas and liver.
is unclear, but may represent expression at higher
levels in a smgl number of specialized cells such as Introduction of the cloned rat elastase I gene into
tissue mast cells (MacDonald et al., 1982b). In yet mice
other tissues such as the parotid and submaxillary
gland, elastase I mRNA is undetectable; expression One approach to test for cell specific expression of
is at least 100,000-fold greater in the pancreas than a cloned gene is to introduce the gene into all cells
in these tissues. The elastase I gene appears maxi- of an animal by microinjection into fertilized eggs
5
Nuclear Run- 1 Transcription
600 . LIVER
500
U
.%-
U
.A- 400
c)r
5
300
0
aI
*N 200
/"
i0 0
_albumin<l ppm e,la stase I c 1p pm
f / f
0 - oI-'--ro ' ' 0 ' 0 -
32P-cprn added ( x ~ O - ~ )
Fig. 2. Pancreas specific transcription of the elastase I gene. Nuclei isolated from rat pancreas and liver were incubated with ["PI
UTP to extend nascedt RNA transcripts. The in vitro transcribed RNA was isolated and hybridized to rat elastase'I and rat albumin
cDNA plasmids bound to nitrocellulose. Background was defined as the amount of hybridization to the cloning vector pBR322. The
relative transcription rates (parts per million) are expressed as the amount of hybridizing counts per minute (cpm) minus background,
divided by the number of input cpm and the length of the cloned cDNA probe. in kilobases (kb). All procedures were performed as
described by McKnight and Palmiter (1979).
to create transgenic animals (Palmiter and Brinster, number of transgenic animal experiments have
1985; Brinster et al., 1985). A major advantage of demonstrated tissue-specific expression of cloned
this method is the ability to assay for expression in genes introduced into mice (Storb et al., 1984; Swift
many tissues of a transgenic animal. Recently, a et al., 1984b; Palmiter and Brinster, 1985). The rat
E P P PHP 2P P EH P P PE EPH H P E
7 8
I I
A I I 111 r'l I )q I
G N K 0 N 0 N K 0 GN N B G N G 0 N B
-6 -4 -2 0 2 4 6 8 10 12 14 16
kb pairs
Fig. 3. The sequence organization of the rat elastase I gene. The positions of the eight elastase I exons within 25 kb of rat nuclear
DNA are shown relative to a restriction map of the region (E, EcoRI; G, BgllI; P, PvuII; N, NcoI; K, @nI; B, BumHI; H, HindII).
The cloned gene region was obtained on two recombinant lambda phage (shown at the top) which contain inserts that abut in genomic
DNA. For microinjection, the gene was reassembled by cloning into pBR322. The gene was prepared for microinjection by cleavage
within the pBR322 vector sequences with CIaI and NruI to create a linear DNA fragment of 27 kb with dissimilar ends. This large
fragment had 25 basepairs of pBR322 sequences at the 5' end and 3.5 kb at the 3' end of the genomic sequences shown.
