Current Topics in Microbiology and Immunology: Ergebnisse der Mikrobiologie und Immunitatsforschung

241 Pages·1969·9.877 MB·English

by  H. G. Schweiger (auth.), W. Arber, W. Braun, F. Cramer, R. Haas, W. Henle, P. H. Hofschneider, N. K. Jerne, P. Koldovský, H. Koprowski, O. Maaløe, R. Rott, H. G. Schweiger, M. Sela, L. Syruček, P. K. Vogt, E. Wecker (eds.)

Current Topics in Microbiology
and Immunology
Ergebnisse der Mikrobiologie und Immunitatsforschung
Volume 50
Edited by
W. Arber, Geneve . W. Braun, New Brunswick . F. Cramer, Gottingen . R. Haas,
Freiburg . W. Henle, Philadelphia . P. H. HoJschneider, Miinchen . N. K. Jerne,
Basel . P. Ko/dovsky, Prague . H. Koprowski, Philadelphia . O. Maalee, Copen
hagen . R. Rott, Giefien . H. G. Schweiger, Wilhelmshaven . M. Sela, Rehovoth
L. Syrueek, Prague· P. K. Vogt, Seattle· E. Wecker, Wiirzburg
With 65 Figures
Springer-Verlag Berlin . Heidelberg . New York 1969
ISBN-l3: 978-3-642-46171-2 e-ISBN-13: 978-3-642-46169-9
DOl: 10.1007/978-3-642-46169-9
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© by Springer-Verlag Berlin· Heidelberg 1969. Library of Congress Catalog Card Number 15-12910.
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accordingly be used freely by anyone. Title No. 4698
Softcover reprint of hardcover 1st edition 1969
Table of Contents
H. G. SCHWEIGER, Cell Biology of Acetabularia. With 20 Figures . 1
S. E. MERGENHAGEN, R. SNYDERMAN, H. GEWURZ, H. S. SHIN, Significance
of Complement to the Mechanism of Action of Endotoxin. With
10 Figures . . . . . . . . . . . . . . . . . . . . . . . . . . 37
O. J. PLESCIA, The Role of the Carrier in Antibody Formation.
With 2 Figures . . . . . . . . . . . . . . 78
A. GRANOFF, Viruses of Amphibia. With 4 Figures 107
A. MAJsKY, Antigenicity of Blood Platelets . . . 138
M. SHILO, Morphological and Physiological Aspects of the Interaction of
Bdellovibrio with Host Bacteria. With 29 Figures 174
Author Index 205
Subject Index 224
Max-Planck-Institut flir Zellbiologie, Wilhelmshaven, Germany
Cell Biology of Acetabularia
H. G. SCHWEIGER
With 20 Figures
Contents
1. Introduction . . . . . . . . . 1
2. Biology ............... . 2
a) Systematics, Occurrence and Description 2
b) Life Cycle . . . . . 3
c) Growth Conditions . . . . 6
d) Classical Experiments. . . 8
e) Morphogenetic Substances. 10
3. Biochemistry . . . . . . . . 11
a) Low Molecular Weight Compounds 11
b) Proteins . 12
c) RNA ......... . 13
d) DNA ......... . 18
4. Semiautonomy of Chloroplasts 19
a) General Considerations . . 19
b) Nuclear Dependent Chloroplast Protein 21
5. Regulatory Mechanisms . . . . . 25
a) Circadian Rhythmicity . . . . 25
b) Regulation of Enzyme Activity . 27
6. Concluding Remarks. 27
References . . . . . . 31
1. Introduction
The various species of the genus Acetabularia are the best known members
of the family Dasycladaceae because of their extensive use as research objects
in the fields of developmental biology, molecular biology and cytology. These
cells combine a number of unique characteristics which make them particularly
well suited for use in these fields. These characteristics include their abnorm
ally large size, their pronounced morphogenetic capabilities and the fact that
they are able to survive for long periods following such operations as enuclea
tion and transplantation of nucleus-containing cell fragments.
1 C.T. in Microbiology. Vol. 50
2 H. G. SCHWEIGER:
The large size is an advantage since it means that a substantial amount
of cytoplasm may be obtained from a single cell. In a number of cases this
has proved to be sufficient for the application of analytical methods to a
single cell. For instance, it has been possible to estimate the enzyme activity
of malate dehydrogenase and the oxygen production during photosynthesis
in single cells. The latter function could be recorded for several days. In
addition, the size of the cell opens up new qualitative aspects: the possibility
of analyzing single cells changes the point of view from a statistical to an
individual one. Questions concerning individual variations from cell to cell
and interrelationships between single cells become accessible problems. Ex
perimental cell biology on the level of single cells represents a promising
approach for a number of problems, such as the effects of nuclear transplanta
tion and the mechanism of circadian rhythms.
Although single cells are usually treated as micro-organisms, the unique
dimensions of a single Acetabularia cell place it in the size range of metazoan
organisms or tissues.
2. Biology
a) Systematics, Occurrence and Description
The systematic classification of Acetabularia has not yet been completely
settled. Most authors regard Acetabularieae as a subfamily of the Dasycladaceae,
order Siphonocladiales (Si phonales) and class Chlorophyceae (O LTMANNS, 1922;
PRINTZ, 1927; FRITSCH, 1965). One of the characteristics of this family is the
tendency towards incrustation with calcium carbonate. Fossil Dasycladaceae
are assumed to have played a major part in the formation of limestone rocks.
The experiments which are to be reported have been performed with
A. mediterranea, A. crenulata, A. wettsteinii, Acicularia schenckii and A. (Poly
physa) cliftonii (Fig. 1). Properties which all members of the subfamily Ace
tabularieae have in common are that they are unicellular and uninuclear
(HAMMERLING, 1931) and that the mature cells consist of rhizoid, stalk and
cap. The rhizoid forms a ball of entangled branches, one of which contains
the cell nucleus. The species specificity of the morphology of the cap provides
a useful means for classification of the different members of the subfamily
Acetabularieae. The maximum diameter of the cap may be 10 mm, and in
some rather uncommon species even more than 10 mm. Likewise the fine
structure of the cell wall exhibits a high species specificity. The stalks of some
of the Acetabularieae may grow to a length of 50 mm or even more. This
property qualifies Acetabularieae to belong to the group of the largest uni
nuclear and unicellular organisms.
Studies of the fine structure of Acetabularia have shown that the greater
part of the cell volume is occupied by a large central vacuole. The cytoplasm
contains the usual sub-cellular organelles, such as chloroplasts (ca. 107 in a
mature cell), mitochondria, Golgi apparatus, etc.
The various members of the subfamily Acetabularieae are found in shallow
waters near the shores of tropical and subtropical seas. The following incom-
Cell Biology of Acetabularia 3
plete list gives the places where different species of Acetabularieae have been
collected:
A. mediterranea: Bay of Naples, Rovinj and Herceg-Novi;
A. crenulata: Bermuda, Bahamas, West Indies;
A. wettsteinii : Bay of Naples;
Acicularia schenckii: Curayao, Bermuda;
A. (Polyphysa) cliftonii: South Japan;
A. calyculus: South Japan.
Fig. 1. Acetabularia cells with caps. From left to right: A. mediterranea, A. crenulata,
A cicularia schenck ii, A. (Polyphysa) wettsteinii, A. (Polyphysa) cliftonii. Actual size
b) Life Cycle
The zygote (Fig. 2b) results from the fusion of two isogametes (Fig. 2a, b).
Within 3 or 4 days a tube-like stalk grows out from one side of the amorphous
zygote and a rhizoid from the other side (Fig. 2c). The rhizoid contains the
single cell nucleus (HAMMERLING, 1931) and is that part of the plant by which
it is attached to the substratum, mostly on stones or rocks. The length of the
stalk is increased by apical growth. In A. mediterranea the growth of the
stalk is associated with the formation of whorls, which are produced at short
intervals along the longitudinal axis of the stalk. The older whorls degenerate
after some time so that only a limited number of whorls can be seen on one
stalk at any given time. Within 3 to 4 months the stalk reaches a length of
30-40 mm and starts to form a cap. In the final stages of development the
diameter of the cap may reach 10 mm. The mature cap exhibits a pronounced
radial design.
During the development of the cell the nucleus undergoes substantial
changes (HAMMERLING, 1931). Until the outgrowth of the zygote, the nucleus
is similar, except for its increased size, to the nucleus of the gamete. At this
1*
4 H. G. SCHWEIGER:
a b
c
Fig. 2. a Gamete from A. mediterranea. b Zygote from A. mediterranea immediately after
copulation. c Outgrowing zygote (x 3,400). By courtesy of Dr. V\,·ERZ
Cell Biology of Acetabularia
point the nucleus starts fo form a nucleolus from which a complex system of
sausage-like structures is finally derived (Fig. 3). The nucleus increases its
size along with the growth of the cell, but at a rate slower than that of the
cytoplasm. The final diameter of the nucleus may be 0.1 mm or more. The
nuclei from mature Acetabularia cells are among the largest nuclei known to
occur.
Fig. 3. Isolated nucleus from A. mediterranea (x 1,000). By courtesy of Dr. VVERZ
When the maximum cap diameter has been reached, the nucleus regresses
and begins to divide in the rhizoid. The secondary nuclei formed by this
process continue dividing mitotically and migrate through the stalk into
the cap. Mitotic division of the secondary nuclei continues during this migra
tory phase. During this time, the cap has taken on a radial design which is
caused by the formation of rays. In A. mediterranea the total number of rays
may reach 100 (HAMMERLING, 1934; SCHULZE, 1939). Up to 250 secondary
nuclei migrate into each ray. Each secondary nucleus is encysted and con
tinues to divide mitotically, and finally undergoes meiosis. More than 2000
gametes per cyst can be formed in this way, thus a cap may contain up to
6 H. G. SCHWEIGER:
107 gametes or even more. The process of cyst formation detailed above is
initiated at the time when the cap reaches its maximum diameter and is
completed within two days.
The formation of cysts is accompanied by a destruction of the cap structure.
At some indefinite time after the release of the cysts, the cysts burst and the
gametes which are now ready for copulation swarm.
The nuclei are diploid during all stages with the exception of the haploid
gametes. In the case of A. mediterranea they contain about 20 chromosomes
(SCHULZE, 1939). The formation of the cap and of the cap chambers ends the
vegetative phase. The cap chambers represent the sexual organs and are
regarded as gametangia by most authors (PRINTZ, 1927). Another rather
plausible account attributes the role of gametangia to the cysts.
c) Growth Conditions
Acetabularieae have been grown in laboratory culture for about 40 yearsl.
Under laboratory conditions morphogenesis in these algae is similar to mate
rial growing in the sea. However, there are some differences in the time needed
for growth. In the sea A. mediterranea, for example, forms cysts not earlier
than two years after copulation, while in the laboratory it matures in about
three months. Another difference is the lack of calcification of the cells grown
in the laboratory. This difference explains why plants in culture look deep
green and the cells in the sea milky green.
Acetabularia cells are grown in "Erd-Schreiber-solution" (HAMMERLING,
1963). Erd-Schreiber-solution is prepared by the addition of an earth decoc
tion to SCHREIBER (1927) solution (PRINGSHEIM, 1954). The earth decoction
is prepared as follows: 100 g of dry garden earth, selected for coarse contam
ination, is autoclaved and then boiled for 90-100 min with 500 ml filtered
sea water. 1710 ml of sea water are boiled for a short time in 2,000 ml Erlen
meyer flasks and 2 days later 45 ml each of NaN03 (4 gil), Na2HP04 X 12 H20
(0,8 gil) and earth decoction are added and the medium is boiled once more.
The exact culture conditions have been described in detail by HAMMER
LING (1944) and BETH (1953) and have been discussed more recently by
LATEUR (1963) and by KECK (1964). The cysts are squeezed out mechanically
from the rays. They can be kept in darkness for months without losing their
capacity to swarm. On the contrary, there is some indication that storage
enhances this capacity. Swarming is induced by illuminating the cysts. The
effectiveness of this induction can be increased by treating the cysts for a few
minutes with distilled water. It is most convenient to have the cysts swarmed
1 In Wilhelmshaven e.g. the following species of Dasycladaceae are being
cultured: 1. Dasycladus clavae/ormis, 2. Batophora oerstedii, 3. Neomeris annulata,
4. Cymopolia barbata, 5. A. mediterranea, 6. A. crenulata, 7. A. calyculus, 8. A. (Poly
physa2) peniculus, 9. A. (Polyphysa) polyphysoides, 10. A. (Polyphysa) cli/tonii,
11. A. (Polyphysa) wettsteinii, 12. A. (Polyphysa) parvula, 13. A. (Polyphysa) clavata
and 14. Acicularia schenckii.
2 The classification of Polyphysa is based on the absence of a corona inferior
in accordance with Solms-Laubach (1895).
Cell Biology of Acetabularia 7
in Boveri dishes. The gametes aggregate on the surface at the side of the dish
towards the light (positive phototaxis) while the zygotes which are formed by
copulation migrate to the opposite side (negative phototaxis) (HAMMERLING,
1934c). Further purification by phototaxis can be obtained by repeated col
lection of the gametes and zygotes and inoculation into fresh medium. Zygotes
which have been obtained in this way are kept in light for one month and can
then be stored in darkness for as long as several years. During storage the
cultures are supplemented with fresh Schreiber-solution every 8th week and
illuminated for five days each time.
The algae are grown in Petri dishes with a diameter of 120 mm, which
contain 100 ml Erd-Schreiber-solution. In every dish 50 plants are grown.
The medium is changed every 14th day. The plants are illuminated with
2,500 lux 12 hours per day. The temperature is approximately 21°C for
A. mediterranea and 24°C for A. crenulata (HAMMERLING, 1963).
The fact that cultures of Acetabularia usually are not free from micro
biological contamination made it difficult to interpret results from incorpora
tion experiments. Therefore the growing of bacteria-free cells of A. mediterra
nea, as described by GIBOR and IZAWA (1963), proved to be highly advan
tageous. Some minor modifications, the application of the method to
A. (Polyphysa) cliftonii and thorough tests for microbiological contamination
have been described by BERGER (1967a). Microbial contamination, mainly
bacteria and molds, is eliminated at the cyst stage, since the cysts are rather
resistant to chemical and bactericidal treatments.
The cysts are incubated for 2 hours in a solution of 10% silver proteinate
(Targesin) at room temperature. The silver proteinate is removed by washing
the cysts five times with sterile sea water. For further treatment the cysts are
kept for 5 days in a solution of 200 mg streptomycin sulfate, 100 mg penicil
lin G, 20 mg neomycin sulfate, 20 mg chloramphenicol, 20,000 international
units of mycostatin and 50,000 international units of bacitracin in 100 ml of
sea water. This solution is passed through a glass bacterial filter before use.
The antibiotics are removed by washing the cysts five times with sterile
90 % sea water. The cysts are shocked by treatment with distilled water for
3 to 5 min to provoke swarming, transferred into sterile medium and illumi
nated (1,200 to 1,500 lux). After swarming and copulation, the resulting
zygotes are collected with Pasteur pipettes and transferred to the test medium.
The zygotes which have germinated are kept in 200 ml Erlenmeyer flasks,
50 plants in each, under light 12 hours per day (1,200 to 1,500 lux). The
medium is changed every 5 to 6 weeks. It is composed as follows: 750 mg
KN0 and 200 mg glycerophosphate are dissolved in 1,000 ml filtered 90 %
3
sea water. This solution is enriched with earth decoction and 2 ml of the
100-times concentrated vitamin mixture of EAGLE. For preparing the test
medium the normal medium is supplemented by 10 mg yeast extract, 20 mg
peptone and 50 mg glucose per 1,000 ml.
The sterility of the cultures is tested by transferring some of the cells
to test medium for 14 days, after which the medium and the plants are ex-