Table Of ContentThe
Chlamydomonas
Sourcebook
A Comprehensive Guide to Biology and Laboratory Use
Elizabeth H. Harris
Chlamydomonas Genetics Center
Department of Botany
Duke University
Durham, North Carolina
Academic Press, Inc.
Harcourt Brace Jovanovich, Publishers
San Diego New York Berkeley Boston
London Sydney Tokyo Toronto
COPYRIGHT © 1989 BY ACADEMIC PRESS, INC.
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ACADEMIC PRESS, INC.
San Diego, California 92101
United Kingdom Edition published by
ACADEMIC PRESS LIMITED
24-28 Oval Road, London NW1 7DX
Library of Congress Cataloging-in-Publication Data
Harris, Elizabeth H.
The Chlamydomonas sourcebook : a comprehensive guide to biology
and laboratory use / Elizabeth H. Harris,
p. cm.
Bibliography: p.
Includes index.
ISBN 0-12-326880-X (alk. paper)
1. Chlamydomonas. 2. Chlamydomonas—Laboratory manuals.
I. Title.
QK569.C486H37 1988
589.4'7-dcl9 88-10453
CIP
PRINTED IN THE UNITED STATES OF AMERICA
89 90 91 92 9 8 7 6 5 4 3 21
To Hannah,
Tommy, and Frieda
Preface
In the Donald Duck comic books I read as a child, the nephews Huey,
Dewey, and Louie belonged to an organization called the Junior Wood-
chucks, which furnished them with a guidebook that provided all neces-
sary information for whatever adventure they were pursuing and had
instructions for resolution of every possible crisis. Lost in the labyrinth,
they had only to turn to the proper page to find a map marked " X" and
"you are here." From the beginning I have envisioned the present book
as the "Junior Woodchuck Guide to Chlamydomonas," a reference tool
of use in all aspects of research on an organism that has become an
important model system for diverse studies in cell biology, genetics, and
biochemistry. Although Chlamydomonas cells are easily cultured and
manipulated in the laboratory, until now there has been no single source
of information on their biology and experimental use. As director of the
Chlamydomonas Genetics Center project, which maintains and distrib-
utes wild-type and mutant strains of the most widely studied species, I
have often been asked for very basic information which I felt ought to be
at hand in every Chlamydomonas laboratory. The book grew out of that
need.
The book begins with an introduction to the genus Chlamydomonas
and to the laboratory strains most commonly used in research. The
second chapter presents methods for culture and storage of Chlamydo-
monas strains and includes tables comparing the composition of the
most widely used culture media. Chapters 3-9 are devoted to reviews of
the major areas of Chlamydomonas biology and research: cellular struc-
ture and the vegetative life cycle; the reproductive cycle; motility and
phototaxis; metabolism; photosynthesis; organelle heredity; and synthe-
sis of nucleic acids and proteins. These chapters are followed by Chap-
ter 10, which describes methods for induction and selection of mutant
strains and their genetic analysis. Chapters 11 and 12 present a compila-
tion of information on the various mutants previously isolated, methods
for laboratory procedures, suggested student laboratory exercises using
Chlamydomonas, and other resources. A comprehensive bibliography is
provided.
The book should be useful to anyone who works with Chlamydo-
monas as a laboratory tool, and I hope that it will encourage among
these scientists something of the "feeling for the organism" described
by Barbara McClintock.
Elizabeth H. Harris
xi
Acknowledgments
Preparation of this book would not have been possible without the help
of a great many people. My colleagues John Boynton and Nick Gillham
supported the project from the beginning, providing me with the time to
work free of other distractions, the wisdom of their experience, and
unfailing encouragement. My first thanks go to them, together with a
promise to get back into the lab and do experiments again.
Ursula Goodenough made the initial contact with Academic Press for
me, reviewed Chapter 4, and searched her files of superb micrographs to
provide many of the figures. I also thank Bert Livingstone and her
library staff, whose speed and efficiency at producing journals were
truly amazing; Judy Reynolds, who kept the copy machine busy;
and Brent Mishler for the use of his microphotography equipment. Fi-
nancial support to the Chlamydomonas Genetics Center by National
Science Foundation Grant PCM-83-09001 is also gratefully acknowl-
edged. Nancy Crisona at Academic Press got the project started, and
Jean Thomson Black and Jean N. Mayer saw it through to completion.
The list of other persons who contributed to the book is almost a
Who's Who of Chlamydomonas biology. For reviews of sections and
helpful discussion, I thank Steve Adair, Mike Adams, Barry Bean, Ka-
ren Brunke, Jacobo Cardenas, David Domozych, Hanus Ettl, Emilio
Fernandez, Charlene Forest, Ken Foster, C. S. Gowans, Bob Hodson,
Bessie Huang, David Husic, Diane Husic, Jon Jarvik, Pete John, Carl
Johnson, Laura Keller, Bob Lee, Pete Lefebvre, Claude Lemieux,
Ralph Lewin, Roland Loppes, David Luck, Renι Matagne, Sabeeha
Merchant, Laurie Mets, Brian Monk, Nicole Morel, Alan Musgrave,
Howard Rosen, Barb Sears, Jim Siedow, Carolyn Silflow, Bill Snell,
Bob Spreitzer, Steve Surzycki, Bob Togasaki, Í. E. Tolbert, Monique
Turmel, Herman van den Ende, Karen VanWinkle-Swift, Andy Wang,
Don Weeks, and George Witman.
Methods sections were contributed by Mike Adams, Bob Bloodgood,
Annette Coleman, Jackie Hoffman, Bob Spreitzer, Bob Togasaki,
George Witman, and Robin Wright. Figures were provided by C. G.
Arnold, C. J. Brokaw, T. Cavalier-Smith, Phillippe Delepelaire, Bill
Dentier, Pat Detmers, Charlene Forest, Å. I. Friedmann, Ursula Good-
enough, C. S. Gowans, Helen Gruber, Jon Jarvik, Pete John, Elspeth
Leeson, Ralph Lewin, Roland Loppes, David Luck, Marjorie Maguire,
Renι Matagne, Alan Musgrave, Kazuo Nakamura, W. Nultsch, Jac-
queline Olive, Jeremy Pickett-Heaps, David Robinson, Jeff Salisbury,
Gregory Schmidt, Martin Spalding, Monique Turmel, Karen
VanWinkle-Swift, Patricia Walne, Dick Weiss, George Witman, and
xiii
xiv Acknowledgments
Robin Wright. The cover design is adapted from a figure supplied by
Hanus Ettl.
During the course of writing I visited the three major algal culture
collections (UTEX, CCAP, and SAG) and several research laboratories.
For their hospitality during these visits I thank Mike Adams, Sue
Bartlett, Ivan and Norma Heaney, Ron and Karen Jacob, George Ja-
worski, Elspeth Leeson, Roland Loppes, Renι Matagne, John Morris,
Uwe Schlösser, Bill Snell, Richard Starr, Bob and Fumiko Togasaki,
Andy and Wanda Wang, and Jeff Zeikus.
Finally, I am grateful to my family for the combination of love, en-
couragement, and growing impatience that kept me working.
1A n Overview
of the Genus
Chlamydomonas
Introduction
This chapter reviews the history of research on species of the genus
Chlamydomonas, with emphasis on the genetically important species C.
reinhardtii and C. eugametos. The origin of the principal laboratory
strains of these species is given in detail (insofar as the historical records
permit), as questions of strain identity may be important in modern
experimental work. The chapter concludes with a brief discussion of
other Chlamydomonas species which have received more than passing
attention in laboratory studies.
Description of the Genus
The genus Chlamydomonas (Greek: chlamys, a cloak or mantle; monas,
solitary, now used as a generic term for certain unicellular flagellates)
was named by C. G. Ehrenberg (1833, 1838), and probably corresponds
to the flagellate Monas described in 1786 (O. F. Müller, cited by Gerloff,
1940; Ettl, 1976a). The species described by Ehrenberg is uncertain; Ettl
(1976a) believes it may have been C. pulvisculus, but since the published
description and illustration could apply to several of the species recog-
nized today, he considers the type genus to be C. reinhardtii, which was
not named until 1888 by Dangeard. The family Chlamydomonadaceae
includes about 800 species in 33 genera, of which Chlamydomonas ac-
counts for by far the greatest number (Bourrelly, 1966; Jakubiec, 1984).
A schematic view of the principal features of a Chlamydomonas cell is
shown in Figure 1.1.
Chlamydomonas is considered by some phycologists to include the
genus Chloromonas, whose cells are similar in overall architecture but
lack pyrenoids. Pascher (1927) combined these two genera in his com-
prehensive treatment of the Vol vocales, but more recent works (Gerloff,
1962; Fott, 1974; Ettl, 1976a, 1983; Prescott, 1978) have usually sepa-
rated them. There is also argument whether Gloeomonas should be
regarded as a separate genus, the principal distinguishing feature of this
group being a slightly wider separation of the flagellar origins compared
1
2 1. An Overview of the Genus Chlamydomonas
Figure 1.1. Schematic diagram of a typical
cell of Chlamydomonas reinhardtii. C, chlo-
roplast; E, eyespot; F, flagella; FR, site of
flagellar roots (see Figure 3.13 for detailed
diagram); G, Golgi; M, mitochondria; N, nu-
cleus with nucleolus; P, pyrenoid; V, vacu-
ole; W, wall.
to those of most Chlamydomonas species (see Ettl, 1965a,c; Fott, 1974).
Another major genus of the same family is Carteria, whose cells have
four rather than two flagella but otherwise look very much like those of
Chlamydomonas. At least one "Carteria" species has been demon-
strated to be a long-lived quadriflagellate product of Chlamydomonas
mating (Behlau, 1939; see Chapter 4), and it is tempting to speculate that
this may in fact be the evolutionary origin of this genus. Sphaerellopsis
and Smithsonimonas have a wide, gelatinous sheath that differs from the
shape of the protoplast, in contrast to Chlamydomonas and Chloro-
monas, in which the sheath, if any, conforms closely to the protoplast
shape. Polytoma is a genus of nonphotosynthetic flagellates that closely
resemble Chlamydomonas in body structure and appear to retain some
vestige of chloroplast nucleic acids and ribosomes (see Pringsheim,
1963a; Siu et al., 1976a-c). The genus Polytomella comprises another
group of colorless species that differ from Polytoma in lacking cell walls.
Dunaliella and related genera form an analogous group of wall-less green
flagellates which in many respects appear very closely allied to the
Chlamydomonadaceae. The evolutionary relationships of Chlamydo-
monas to other genera, and particularly the position of the Volvocales as
a side branch in the development of higher plants from green algae, have
been explored in detail by investigators in several laboratories. The
composition and organization of the cell wall, the morphology of the
flagellar root system, and the structures involved in cytokinesis are the
most significant features contributing to evolutionary schemes within
Description of the Genus 3
the Chlorophyta (see Kochert, 1973; Pickett-Heaps, 1975; Ettl, 1981; see
also Chapter 3 for further discussion).
Although cell body shape and size vary among Chlamydomonas spe-
cies (Figure 1.2), the overall polar structure, with paired apical flagella
and basal chloroplast surrounding one or more pyrenoids, is constant.
Cells are usually free-swimming in liquid media but on solid substrata
may be nonflagellated and are often seen in gelatinous masses similar to
those of the algae Palmella or Gloeocystis in the order Tetrasporales.
This condition is referred to as a "palmelloid" state (Fott and Nov-
akova, 1971). There is even some discussion that Gloeocystis may com-
prise palmelloid Chlamydomonas species for which no motile stage has
been identified (Badour et al., 1973). The Chlamydomonas wall is dis-
tinct, with some variation in thickness among species, and some species
secrete a mucilaginous polysaccharide coating. Most species have a
prominent eyespot, usually red, and two or more contractile vacuoles.
Asexual division occurs by lengthwise division of the protoplast. Usu-
ally two successive divisions occur to form four daughter cells, which
are then released from the mother cell wall. The forms of sexual repro-
duction range from isogamy (fusion of morphologically similar gametes,
the most prevalent form; Figure 1.3) to oogoniogamy or oogamy (forma-
tion of clearly differentiated egg and sperm cells) and are not diagnostic
of the genus (see Chapter 4 for further discussion). For many of the
described species, no sexual cycle has been observed. Sexual fusion of
whatever style leads to formation of a diploid zygospore with a hard,
thick wall, which is resistant to adverse environmental conditions. Some
species also form asexual resting spores, or akinetes.
Dill (1895) listed 15 species of Chlamydomonas, of which six were
new descriptions. By 1927 the list had grown to 146 species found in
central Europe. Pascher (1927) delineated six subgenera based on chlo-
roplast shape and number and position of the pyrenoid(s), and Gerloff
(1940) provided a new key to these and described additional species,
bringing the total to 321. The most recent comprehensive work on the
genus is Ettl's (1976a) monograph, in which the literature on 459 species
is summarized. Ettl elevates the previous subgenus Chloromonas to a
separate genus and divides the remaining species into nine groups,
which he prefers to call Hauptgruppen rather than subgenera, implying
no formal taxonomic rank (Figure 1.4; Table 1.1). Apart from his assign-
ment of all snow- and ice-dwelling forms to one group (Sphaerella), Ettl
considers neither habitat nor mode of reproduction in making these
major divisions. Within most of the nine groups, Ettl further separates
species with similar chloroplast morphology. Species are distinguished
from one another by several traits, including presence or absence of a
pronounced apical papilla, number and position of contractile vacuoles,
overall body shape, thickness of the cell wall, shape and position of the
eyespot, and whether a gelatinous sheath surrounds the cell. The varia-
tions in chloroplast shape within the genus, and a possible scheme for
4 1. An Overview of the Genus Chlamydomonas
Figure 1.2. Diversity of body shapes within the genus Chlamydomonas. (A) Chlamydo-
monas incerta; (B) C. biconvexa; (C) C. venusta; (D) C. penium; (E) C. diffusa; (F) C.
basimaculata; (G) C. perpusilla; (H) C. lagenula; (I) C. lunata; (J) C. gyroides; (K) C.
conica; (L) C. musculus; (M) C. svitaviensis; (N) C. lismorensis; (O) C. longeovalis\ (P) C.
tetragama; (Q) C. chlorogonioides\ (R) C. spinifera; (S) C. citriformis; (T) C. constricta;
(U) C. incurva; (V) C. depressa; (W) C. chlorogoniopsis; (X) C. formosissima; (Y) C.
ranula; (Z) C. opulenta; (AA) C. bergii; (BB) C. curvicauda; (CC) C. rhinoceros; (DD) C.
complanata, with cross-sectional view below; (EE) C. dorsoventralis; (FF) C. securis.
From Ettl (1976a).
