Download isolation and genetic analysis of mutant strains in gametic

ISOLATION AND GENETIC ANALYSIS OF MUTANT STRAINS
OF CHLAMYDOMONAS REZNHARDZ DEFECTIVE
IN GAMETIC DIFFERENTIATION
URSULA W. GOODENOUGH, CAROL HWANG
AND
HOWARD MARTIN
The Biological Laboratories, Harvard Uniuersiiy, Cambridge, Massachusetts 02138
Manuscript received February 10, 1975
Revised copy received October 22, 1975
ABSTRACT
Impotent mutant strains of Chlamydomonas reinhardi, mating-type ( m t )
plus, are described that have normal growth and motility but fail to
differentiate into normal gametes. Procedures for their isolation and their
genetic analysis are described. Five of the imp strains (imp-2, imp-5, imp-6,
imp-7, and imp-8) exhibit no flagellar agglutination when mixed with mt- or
mt+ gametes; these strains have been induced to form rare zygotes with mtgametes and the mutations are shown to be unlinked to the mi locus (with the
possible exception of imp-7).Two of the strains (imp-3 and imp-4) carry leaky
mutations that affect cell fusion; neither mutation is found by tetrad analysis
to be linked to mt or to the other. Cells of the imp-I strain agglutinate well
with m t gametes and active agglutination continues for up to 48 hours, but
cell fusion occurs only very rarely. Analysis of these rare zygotes indicates that
imp-1 is closely linked to the mt+ locus, and fine-structural studies reveal that
imp-I gametes produce a mutant mating structure involved i n zygotic cell
fusion. The development of sexuality in C. reinhardi therefore appears amenable to genetic dissection.
HE unicellular biflagellate green alga Chlamydomonas reinhardi is often
Tcited as representing the kind of eukaryotic protist that once gave rise to the
multicellular plants and animals: it possesses all of the “standard” organelles
1957; RINGO1967; JOHNSONand
found in metazoan cells (SAGERand PALADE
PORTER
1968), but it has not acquired the exotic structural features found in the
more highly evolved protozoa; its chloroplast membrane polypeptides (LEVINE
and DURAM1973) and chloroplast functions (LEVINE1968) are similar to a
higher plant such as spinach; its chromatin structure and mitotic behavior
resemble higher organisms and do not exhibit the “deviations” found in such
organisms as yeast (MOENS
and RAPPORT1971) or dinoflagellates (KUBAIand
RIS 1969); and its haploid DNA content [0.1 picogram (CHIANG and SUEOKA
1967)] is of the same order of magnitude as an organism like Drosophila r0.25
picogram (MULDER,
VAN DUISINand GLOOR
1968)l. If such a central evdutionary position is supposed, then it becomes of considerable interest to investigate
the genetic and biochemical capacity of C. reinhardi to carry out cellular differentiation and to establish cell-to-cell contacts, both kinds of processes being
clearly critical to metazoan existence.
Genetics 82: 169-186 February, 1976
170
U . GOODENOUGH, C. H W A N G A N D H . M A R T I N
Cellular differentiation in the C. reinhardi life cycle is most dramatically
undertaken when nitrogen is removed from the medium and vegetative cells of
the two mating types (mt+ and mt-) differentiate independently into gametes
(SAGERand GRANICK
1954). Gametes of opposite mating type are uniquely able
to establish specific cell-to-cell contacts via their flagella and to fuse with one
another. Because this gametic differentiation can be induced to occur both rapidly
(12-15 hours) and synchronously (KATESand JONES1964),it has been subjected
to a number of fine-structural and biochemical studies (KATESand JONES 1964;
WIESE1969; SIERSMA
and CHIANG1971; SCHMISSER,
BAUMGARTEL
and HOWELL
1973; SNELL,KROOPand ROSENBAUM1973; GOODENOUGH
and WEISS1975;
MARTINand GOODENOUGH
1975; BERGMAN
et a2. 1975). Lacking, however,
has been a collection of mutant strains defective in the mating process analogous
to those isolated for yeast (HAWTHORNE
1963; MACKAY
and MANNEY
1974a and
1942, 1974; SONNEBORNand LYNCH1937;
b) , Paramecium (SONNENBORN
TAUB1963; BYRNE1973), and Volvox (SESSOMS
and HUSKY
1973).
The present paper reports the isolation and characterization of eight mutant
strains of C. reinhardi that are unable to undergo a normal gametogenesis and
are designated impotent (imp).The strains were selected to express their phenotypes at room temperature. Temperature-sensitive strains were not sought for
several reasons: (1) OUT screening procedure proved, in pilot experiments, to be
most effective at 25"; (2) all available information on gametogenesis derived
from studies performed in the 22"-26" temperature range and it seemed preferable to analyze our first strains in the context of these prior experiments; and
(3) we were able to induce most of the strains to form rare zygotes that could be
recovered and analyzed; therefore, their non-conditional nature did not appear
to preclude their genetic characterization. Unfortunately, the unsuspected presence of non-selective lethal genes in the parental strain has limited the kinds of
genetic information we can obtain from these zygotes. Nonetheless, the present
report serves to establish that sexual differentiation in C. reinhardi is amenable
to genetic analysis, that a minimum of four gene loci are involved in gametogenesis, that at least three loci are unlinked to mating type, and that one locus is
closely linked to, and may lie within, the mt+ locus.
MATERIALS A N D METHODS
Strains and growth conditions
The wild-type C. reinhardi strain 137c-H mt+ was used as the parent strain for all the
mutant strains isolated. Its properties are described in a later section of the paper. The wild-type
strain 137c-H m t , the arginine-requiring strain arg-2 mt-, and the acetate-requiring strain,
ac-29 mt- (provided by N. W. GILLHAM)
were used in various crosses.
and LEVINE1965)
Cells were generally grown on Tris-acetate-phosphate (TAP) (GORMAN
medium solidified with 1.5% agar (Difco) ; this was supplemented with 10 pg arginine per ml
for the arg-2 strain. Solid media for zygote maturation were prepared by adding agar (4%) to
TAP or arginine-supplemented TAP. Synchronous cultures (KATESand JONES1964.; MARTIN
CHIANG
and GOODENOUGH
1975) were grown in high salt minimal (HSM) medium (SUEOKA,
and KATES 1967) and gametogenesiswas induced in nitrogen-free (N-free) HSM.
GAMETIC DIFFERENTIATION MUTANTS
171
Cells were grown on solid media in glass Petri dishes at constant temperature (25") in
continuous light (3600 lux) from daylight fluorescent lamps; they were transferred to fresh
media weekly. Vegetative cells were grown asynchronously in liquid TAP medium; they were
also grown synchronously in liquid HSM on a 12-hour light/le-hour dark cycle as described
elsewhere (MARTINand GOODENOUGH
1975). Synchronous gametes were obtained by harvesting such synchronous vegetative cultures during the sixth hour of the third synchronous
cycle (KATESand JONES1964), resuspending the cells into N-free HSM, and maintaining them
1975). Plate gametes were
in continuous light for 15 hours (MARTINand GOODENOUGH
obtained by inoculating N-free HSM (supplemented, when appropriate, with acetate or arginine)
with cells that had been growing on solid media for at least 5 days; such cells have differentiated
1975) and require only about an hour
into gametes on the plates (MARTINand GOODENOUGH
in liquid to disperse and grow flagella before acquiring full mating efficiency.
Determination of mating efficiency
Mating efficiency was determined by mixing equal numbers of mt+ and mi- gametes and
determining the reduction in cell number caused by zygotic cell fusion after a 1% hr period.
Details are given elsewhere (CHIANG
et al. 1970; MARTIN
and GOODENOUGH
1975). Cell number
was estimated using a hemocytometer.
Mutagenesis
Vegetative cells were grown overnight in liquid TAP medium and were adjusted to a
concentration of 5 x 106 cells/ml in a total of 20 ml of TAP (all manipulations were performed
using sterile conditions). Four-ml aliquots were then transferred t o small Petri dishes containing
magnetic stirring bars. While being stirred vigorously, the suspensions were exposed to ultraand LEVINE1962), a dose that
violet (UV) light (97 ergsan-*,sec-1) for 45 sec (GILLHAM
produces a 90% kill. The aliquots were then transferred to a common flask and a second flask
was prepared containing equal numbers of nonirradiated control cells. Both samples were placed
in the dark for at least 15 hours to prevent photoreactivation and were allowed to undergo at least
one round of mitosis, determined as a doubling of the control cell number, to allow UV-induced
damage to elicit permanent mutations. The approximately 1 x 106 viable cells/ml were then
returned to the light.
Screening f o r non-mating gametes
The UV-irradiated and the control mt+ samples were each spun down and resuspended in
2 ml of N-free HSM. Both samples were left in the light until the control cells exhibited near
100% mating efficiency (usually overnight). Two rounds of mitosis normally accompany
gametogenesis, so that an estimated 2 4 x l o 7 viable gametes were present in the 2 m l of
irradiated sample, an estimate confirmed by plating an aliquot of the sample and counting the
number of colonies. These m t f gametes were spun down and resuspended in 0.5 ml, to which
0.5ml of N-free HSM was added containing approximately 8 x 107 gametes of arg-Zmt-,
previously prepared and ascertained to mate with near 100% efficiency. The resulting mating
mixture was left in the light overnight, allowing a thick, self-adhesive pellicle of zygotes to
form at the meniscus and to adhere to the sides of the test tube. The pellicle was carefully
removed and the liquid, presumably containing only the excess arg-2 cells and any m t f cells
that have failed to mate, was slowly poured into an empty test tube. Aliquots containing 0.1 ml
of this liquid were spread on the surface of 1.5% agar-TAP plates, a medium that disallows
growth of unmated arg-2 mi- cells. Each viable unmated mt+ cell formed a vegetative colony;
in a typical experiment, 100-300 colonies appeared per plate. Individual colonies were picked,
replated, and maintained in culture while their mating capacities were tested. Clones that were
unable to form zygotes with m t - gametes were then examined microscopically for flagellar
motility; their ability to mate with m t f gametes was also investigated.
172
U . GOODENOUGH, C. H W A N G A N D H. M A R T I N
Mating and genetic analysis of mutant strains
For crosses involving imp-1, imp-3, and imp-4, matings were performed by mixing imp and
mi- plate gametes in a small tube for 2 hr (imp-3 and i m p 4 or overnight (imp-1) and then
spreading 1 ml aliquots on 1.5% or 4% agar-TAP plates. For crosses involving imp strains,
imp and m e plate gametes were mixed for either 1 hr or overnight. They were then centrifuged
together at 800 x g €or 2 h r at room temperature (a manipulation that has no adverse effect on
mating between wild-type gametes), followed by spreading 1 ml aliquots on 1.5% agar-TAP
plates.
In all cases, the plated cells were exposed to light for 24 h r and maintained in darkness for
5 days to allow zygote maturation (EBERSOLD
and LEVINE1959). For i m p 3 and imp4 crosses,
zygotes were recovered and subjected to tetrad analysis by conventional procedures (EBERSOLD
and LEVINE1959). For the remaining matings, where zygotes are too rare to be collected and
manipulated, the plates were returned to the light, the overlying growth of vegetative cells was
gently scraped to the sides of each dish with a sterile razor blade, and each plate was everted
over a dish containing chloroform for 3 0 4 0 seconds. A 15-second chloroform exposure was
determined to kill all vegetative cells in control experiments, whereas the thick-walled zygotes
and LEVINE
1959). [MCVITTIE(1972) has independently
survive the chloroform (EBERSOLD
taken advantage of centrifuging and of chloroform treatment to isolate rare zygotes resulting
from crosses involving short-flagellum mutants.] The chloroformed plates were then left i n the
light for 1-2 weeks, allowing any zygotes present to form a zygotic colony made up of mitotic
clones of its viable meiotic products.
To analyze the phenotypes of the constituent cells in each zygotic colony, a dilute suspension
of each colony was prepared and spread on the surface of an agar plate. Sixteen of the resultant
colonies, termed zygotic clones, were randomly picked for further analysis. Gametic cells from
each clone were mixed with tester mt+ and mt- gametes to determine mating type and ability
to form a zygotic pellicle. In the case of matings involving the arg-2 or ac-29 strains, the arginine
or acetate requirements of each clone were determined by plating to unsupplemented media.
RESULTS
Yield and general characteristics of mutant strains
The screening procedure for isolating non-mating gametes, described in detail
in MATERIALS AND METHODS, depends on the fact that zygotes secrete an adhesive
substance and so stick together in a self-adherent pellicle that can be removed
from a mating mixture, leaving unmated and potentially mutant cells behind.
Since an estimated 1-2 X lo6 viable mt+ are initially present in each 0.1 ml of
the mating mixture and since approximately 2-3 X lo2 mt+ cells are recovered
after mating and pellicle formation are allowed to occur, it is clear than an
approximate 10,000-fold enrichment for unmated cells is achieved by removing
the pellicle of zygotes.
Of the 100-300 colonies per plate that remain, however, roughly 98% prove
capable of normal mating when retested. Some of these “spoilers” may have been
subject to the obscure “physiological incompetence” described by CHIANGet al.
(1970) rather than from any heritable genetic defect; in this regard we should
stress that near-100 % mating efficiency is essential in this screening procedure
if such “spoilers” are not to completely swamp out true mutant strains. Other
“spoiler” colonies may derive from zygotes that fail to join the pellicle.
The screening procedure has thus far yielded 16 mutant strains representing
three distinct phenotypic categories. (1) Eight strains carry mutations that
GAMETIC DIFFERENTIATION MUTANTS
173
appear to affect gametic differentiation directly; these are designated impotent
(imp) and are considered in detail in later sections. (2) Seven strains carry
mutations that prevent the formation of flagella, resulting in an inability to
undergo the flagellar-agglutination reaction that serves as a prelude to mating;
these are designated bald and their properties are described in part elsewhere
(GOODENOUGH
and ST. CLAIR1975). (3) One strain carries a mutation that
affects its overall physiological state: specifically, it grows well on agar medium
but swells and becomes moribund in N-free minimal medium. The general
absence of such disabled strains can probably be attributed to the nature of the
screening procedure: the colonies that emerge have had to survive at least 3 days
of growth in liquid medium and several weeks of growth on solid medium following UV-irradiation; moreover, care was taken to avoid picking colonies for
analysis that seemed small and/or pale.
Aberrant property of the parental 137c-H strain
Before describing the phenotypic traits and genetic analyses of the eight
impotent strains we have isolated, it is essential to report a property of the parent
strain from which these mutants derived. The original wild-type strain 137c was
used extensively in the laboratory of R. P. LEVINEat Harvard University for
genetic analysis (EBERSOLD
et al. 1962) and exhibited no unusual properties. In
1968, mt+ and mt- cells of this strain were obtained from the Harvard laboratory
by R. K. TOGASAKI
and taken to Indiana University, where they were maintained
in culture. Subsequently, DR. TOGASAKI
sent us cells from his cultures (which
we propose to designate 137c-H mt+ and 137c-H mt-). Both groups of investigators proceeded to use these as parent strains for the isolation of various classes
of mutants (HUDOCK
and TOGASAKI
1974), and both independently undertook
genetic analysis of their mutant strains.
Subsequent communication between the groups has revealed that the 137c-H
strains contain loci that inflict lethality on meiotic products. For example, in this
laboratory, the cross 137c-H mt+ X arg-2 mt- was performed and 14 zygotes
were analyzed that contained four meiotic products each. Ordinarily, all four
products are viable on arginine-supplemented medium (4/4 viability). I n this
case, however, only seven of the 14 zygotes exhibited 4/4 viability; of the seven
remaining zygotes, one exhibited 3/4, two exhibited 2/4, and four exhibited 0/4
viability. Examination of the plates with a dissecting microscope revealed that
some non-viable products had undergone one or two divisions before dying, while
others had failed to divide at all, Similar degrees of mortality were observed in
cases where the four meiotic products had undergone one or two mitoses before
the zygote wall was ruptured. The fact that all four (or all eight) of the germinating cells frequently die suggests that lethal events may often occur in the
zygote cytoplasm prior to meiotic cytokinesis.
Analysis of the survivors in the above cross revealed no selective lethality for
mt+ over mt- cells nor for arg over prototropic cells. Similarly, in crosses using
the imp strains, imp and wild-type products were found to be equally susceptible
to the lethal effects, as documented in Table 1 for crosses involving imp-3 and
174
U. GOODENOUGH, C. HWANG A N D H. MARTIN
TABLE 1
Genotypes of meiotic products inviable during early germination of
zygotes deriving from strain 137c-H
Cross
Tetrad
Viability
x
D2
214
D3
D5
3/4
214
D7
D9
3/4
214
D18
2/4
e5
C6
3/4
2/4
C13
E10
E13
3/4
3/4
2/4
imp-3 mt+
i m p 4 mt+
x
+ mt-
+ mt-
F2
Inferred genotype(s) of
inviable product(s)
+ mt+
++ mtmt+
imp-3 mimr
imp-3 mt+
imp-3 mtf mtmt+
i m p 3 mt+
mimtf
i m p 4 mt+
imp-4 mtimp-4 miimp-4 m t f
imp-4 mt+
imp-4 mt+
mt+
imp4 mtf
f mt+
mimt+
imp-4 mt+
+
+
++
+
F3
F7
3/4
GI
G6
GI 0
3/4
3/4
3/4
7/8
++
imp-4, the two imp strains that can be subjected to tetrad analysis (see below).
To compile this table, the total number of cells emerging from each zygote was
recorded, the genotypes of the colony-forming cells were determined, and the
genotypes of the inviable cells in each zygote were inferred assuming normal
allelic segregation (normal segregation occurred in all of the zygotes exhibiting
4/4 viability).
The lethal process presumably explains the fact that of the rare zygotic colonies
isolated by the centrifuge-chloroform procedure described in MATERIALS AND
METHODS, about 25% prove to contain cells of only one genotype. That such
colonies drive from zygotes and not from unmated cells that survived chloraforming is indicated by the totally effective killing of unmated cells by chloroform in control experiments and by the fact that the cells in such colonies were
frequently found to exhibit a single recombinant genotype.
The occurrence of such lethal events clearly limits the information that can be
derived from the analysis of rare zygotes. For example, a colony containing two
parental types might represent a parental ditype or it might represent a tetratype
where the two recombinant products have not survived. We therefore present
GAMETIC DIFFERENTIATION MUTANTS
175
our rare zygote data without attempting to classify zygotes as parental, tetratype,
or nonparental ditype.
General properties of the impotent mutant strains
It is well known in Chlamydomonas genetics laboratories that, for example,
mutant strains with impaired photosynthesis (LEVINE
and GOODENOUGH
1970)
tend to be reluctant maters; certain mating-defective strains of Paramecium
apparently exhibit growth anomalies as well (SONNEBORN
1974). Extensive
studies on the growth properties of the eight impotent mutant strains that have
been isolated indicate that their inability to mate is not due to any detectable
defect in their general physiology. All have normal chlorophyll levels, normal
flagellar length and motility, normal cytoplasmic fine structure, and all grow at
wild-type rates both on 1.5% agar-TAP plates and in synchronous culture in a
minimal salts medium (HSM) .
When induced to undergo gametogenesis in synchronous culture, all of the imp
strains undergo the expected mitosis at the end of the 12-15-hr differentiation
period (CHIANGet al. 1970), and all are found to acquire many of the finestructural features of competent gametes (MARTINand GOODENOUGH
1975).
I n the sections that follow, each strain or group of strains is considered in terms
of specific phenotype and of behavior in crosses.
Non-agglutinating impotent strains
The imp-2, imp-5, imp-6, imp-7, and imp-8 strains were each isolated in independent experiments but all exhibit a similar phenotype: none will undergo
flagellar agglutination with mt- (or normal m t f ) gametes, and none will form
zygotes with mt- cells unless centrifuged (see MATERIALS AND METHODS).
Table 2 summarizes the analyses that have been performed of such zygotes.
Each imp strain was mixed with m t gametes either briefly (Type A cross in
Table 2) or overnight (Type B cross) ; it is clear from Table 2 (column 3) that
greater numbers of zygotic colonies are produced by Type B crosses, an enhancement resulting from a gradual loss of cell walls during the overnight suspension
(see GOODENOUGH
and WEISS 1975), which improves the probability of cell
fusion during the subsequent centrifugation. Two additional features of the
crosses are apparent from column 3 of Table 2. (1) The frequency of zygote
formation is very low: approximately IO7 cells are applied to a plate following
a centrifuge mating, yet the average number of zygotic colonies per plate ranges
irom 0-75 (discounting the second imp-2 cross in which limited reversion may
have occurred). (2) Success in zygote formation varies with the strain, the
relative order of success being imp4 >imp-Z>imp-&>imp-5
>imp3 (again
discounting the second imp-2 cross).
The zygotic clones (MATERIALS AND METHODS) that emerge from each zygotic
colony are of three possible genotypes: mt- (mates with mt+ tester gametes),
mt+ (mates with mt- tester gametes), or imp (no agglutination or mating;
mating type therefore unknown). Table 2 indicates, for a given cross, the number
of zygotic colonies that contained clones of particular geEotypes (e.g. for the first
+
+
176
U. G O O D E N O U G H , C. H W A N G A N D H. M A R T I N
TABLE 2
Genetic analysis of strains imp-2, imp-5, imp-6, imp-7 and imp-8
Total
no.
zygotic
zygotic
colonies colonies
per plate analyzed
Average
no.
Cross
imp-2mtf
imp-5 mt+
x
+m t
x fm
t
+ mtimp-7 mtf X + mtimp-8 mtf X + mtimp-6 mtf X
Type of
mating'
A
B
A
B
A
B
A
B
A
B
5
loot
0
4
0
20
0
0.5
25
75
Genotypes of zygotic clones found in zygotic colonies
+andmi-
mt+, + mt+ + mi+
++ mt-,
+ mt- andimp
+andmt- and
imp
+ mt+
imp
imp
14
14
4
3
1
0
2
0
2
4
0
6
2
1
3
0
13
5
1
1
0
5
1
0
29
7
2
5
5
8
2
0
2
32
18
1
6
2
0
2
1
1
0
3 1 3
4
4
0
4
4
0
0
4
0
2
1
* A, gametes mixed, centrifuged 2 hr, germination plates chloroformed; B, gametes mixed
overnight, then treated as A.
The large total number of colonies and the relative abundance of fm t f ,
m t colonies
suggests that some of the cells in this cross carried a +mt+ gene, either uin suppression or
reversion. In two years of culture, this represents the one likely occurrence of reversion or
suppression of an imp strain.
+
+
+
cross involving imp-2, four zygotic colonies contained mt- and imp clones, one
colony contained only imp clones, and so on). Clones of the genotype mt+ will
be expected to occur frequently if a particular imp locus is either loosely linked or
unlinked to the mt locus, and it is clear from the last four columns of Table 2
that zygotic colonies containing mt+ clones are common in the case of imp-2,
imp-&,and imp-8 crosses. They also occur in crosses involving imp-5, although
the data are less convincing because five of the six zygotes are of the mt+,
4-mt- variety which could have arisen by rare repression or suppression events.
N o + mt+ clones have been fpund f o r imp-7, but since only two zygotic
colonies have ever been recovered from this strain despite repeated attempts, no
significance can be attached to this result.
+
+
+
Non-fusingstrain imp-i'
The imp-1 strain was isolated independently of all other imp strains and
exhibits a unique phenotype. When imp-1 is mixed with mt- gametes, an
apparently normal gametic agglutination takes place; however, the agglutinating
clumps continue to grow in size until the cells visibly settle to the sides and
bottom of the test tube in huge clusters, and agglutination continues vigorously
for at least 48 hrs without any apparent zygote or pellicle formation. I n contrast,
wild-type gametes fuse to f o m zygotes within 5-10 minutes; since zygote
flagella are somehow rendered non-agglutinable at the moment of cell fusion
i WIESE1969), the large clumps that initially form between wild-type gametes
rapidly diminish in size as zygote formation ensues.
GAMETIC DIFFERENTIATION MUTANTS
177
178
U. GOODENOUGH, C . HWANG A N D H. MARTIN
Although no apparent zygotic fusion occurs in an imp-lx mt- cross, rare
zygotes can be recovered without centrifugation, indicating that the mutation is
slightly leaky. Table 3 summarizes the clonal analysis of 60 such zygotes. In
marked contrast to the results with the non-agglutinating imp strains (Table 2 ) ,
no recombination had occurred between imp-1 and mt+: of 960 zygotic clones
analyzed, no mt+ or imp-l mt- clones were recovered from any of the zygotic
colonies.
To determine whether this close linkage ex tended to other markers located
near mt, the cross imp2 mt+ x mt- ac 29 was performed (with centrifugation), ac-29 being tightly linked to mt (GILL.HAM
1969) and available in a minus
strain. Zygotic colonies were isolated and cloned just as soon as colonies became
visible on the plate to prevent ac-29 products from being numerically overwhelmed by the faster-growing prototrophs. The clones were then replica-plated
to upsupplemented media and 16 acetate-requiring and 16 acetate-independent
colonies were picked from each zygote. All the acetate-requiring clones were
f o m d to be mt- and to mate normally, whereas all the prototrophic clones were
mt+ and exhibited the imp-1 phenotype (Table 3 ) . Therefore, the imp-1 locus
is clearly linked to both the mt and the ac-29 loci.
Finally, we performed two crosses to determine whether the apparent linkage
between imp1 and mt+ was in fact the result of some generalized inhibition of
recombination or independent assortment conferred by the imp-1 mutation. As
is seen in Table 3, a free assortment of genes occurs when the third marker in the
cross is either imp4 (linkage group undetermined) or arg-2 (linkage group I).
'Therefore, the lack of recombination between imp-1,ac-29, and mt+ (all linkage
group VI) does not extend to other unlinked markers in a zygote.
+
+ +
Pmrly fusing strains imp-3 and i m p 4
Strains imp-3 and imp-4 were isolated in the same mutagenesis experiment and
exhibit similar phenotypes: both strains agglutinate normally with mt- gametes
but fuse with low efficiency. This efficiency varies inexplicably: in three separate
eztimates where control wild-type cells mated with 100% efficiency, imp-3 cells
mated with 12%, 26%, and 50% efficiency. The reduced efficiency is not caused
by there being a mixture of normal and mutant genotypes in the strain since
numerous mitotic clones derived from individual imp-3 and imp-4 cells all exhibit
low-efficisncy matings. A fip.al characteristic feature of the imp-3 and imp-4
phenotype is that the unmated gametes in a mating mixture are still agglutinating strongly to mt gametes 12 hours after mating is ipitiated. This trait
distinguished an imp-3 or imp-4 mating from a low-efficiency mating involving,
for example, a physiologically disabled mutant strain, and is a trait shared with
the mutant strain imp-2 (see precedicg section).
The leakiness of imp-3 and imp-4 has permitted the recovery of zygotes by
conventional methods and their characterization by tetrad analysis. Results of
these crosses are summarized in Table 4. It is evident that neither mutation is
linked to the mt locus (linkage group VI) (see also Table 3 ) nor to the arg-2
locus (linkage group I).
179
GAMETIC DIFFERENTIATION M U T A N T S
TABLE 4
Tetrad analysis of strains imp-3 and imp-4
Total scorable zygotes’
Cross
+
+ +
+
+ +
imp-3 mt+ x
miimp-3 X
arg-2
imp-4 mt+ x
mtimp-4
x arg-2
PD
T
NPD
2
21
2
5
25
9
3
3
1
26
10
27
14
1
0
5
* A scorable zygote had at least two out of four viable products such that the genotypes of the
inviable products could be inferred.
The high frequency of tetratypes exhibited by imp-3 and imp-4 is noteworthy.
The fact that both strains have similar phenotypes, were isolated in the same
mutagenesis experiment, and exhibit high recombination frequencies in crosses
led us to question whether we had isolated two mitotic representatives of the same
mutant cell. W e therefore performed an imp-3 mt+ x imp4 mt- cross and
analyzed the resulting zygotes by the “rare zygote” procedure. Zygotic clones
could only be classified as wild-type o r impotent, it being impossible to distinguish
imp-3 from imp-4. Of eight zygotes analyzed, four contained both wild-type and
impotent clones (Table 5 ) , suggesting that the two mutations are not allelic,
although the wild type were all mt-,an outcome we cannot readily explain.
We also performed an imp-3 mt+ x imp-3 nit- cross to determine whether the
strain might carry two unlinked mutations that were assorting independently to
give a high yield of apparent tetratypes. Of seven zygotes analyzed, however,
none contained the wild-type clones that would be expected to emerge if the
imp-3 strain were doubly mutant at unlinked loci.
Finally, we performed the cross imp4 mt+ x mt- arg-2 to determine
whether the excess of tetratypes would extend to a third marker in the zygote. We
found a tetrad ratio of 1 PD: 13 T:O NPD when mt arrd imp4 were considered
and a ratio of 0 PD:9 T:5 NPD when nr,a and imp-4 were considered, again
+ +
TABLE 5
Genetic analysis of zygotes resulting froman imp-3 mt+ X imp-4 mt-cross
Zygote
1
2
3
-
4
-
5
6
7
8
+
+ mt+
-
+ mt-Genotype of clone*
imp mt+
-
++
+
+
-
++
++
+++
imp m f -
+
++
++
+
-
* indicates that at least one clone of that genotype was present in the zygote - indicates
none was present.
180
U. GOODENOUGH,
C. H W A N G
A N D H. MARTIN
revealing a great excess of recombinant tetrads with respect to the imp-4 marker.
I n contrast, a ratio of 5 PD: 7 T:2 NPD was observed when mt and arg were
considered, indicating that the presence of the imp-4 mutation was not causing
excessive rates of recombination between m t and arg. Similar statistics were
X 4- mt- arg-2
obtained when ten zygotes were analyzed from an imp-3 mt+ icross.
Therefore, our results lead us to conclude that imp-3 and imp-4 are two
unlinked or distantly linked genes and that each occupies a position on its
respective chromosome that experience high rates of recombination (e.g., a
position very far removed from the centromere).
DISCUSSION
Genetic analysis of unconditional impotent strains
Unconditional impotent m t + cells, particularly those that cannot undergo
flagellar agglutination, must be induced to lose their cell walls and then centrifuged together with mt- gametes in order to obtain an appreciable yield of
zygotes. Presumably the centrifugation brings into occasional contact the anterior
COLWINand COLWIN
membrane regions specialized for cell fusion (FRIEDMANN,
1968; GOODENOUGH
and WEISS1975), regions that we have recently determined
by freeze-cleave electron microscopy to be free of intramembranous particles and
hence particularly prone to fuse with one another (WEISS,GOODENOUGH
and
GOODENOUGH,
manuscript in preparation). The rare zygotes that result must
then be recovered by killing all unmated cells, and the zygotic products that
emerge must be analyzed by cloning. While such procedures have been shown
here to yield genetic information, they are long and tedious and virtually
preclude those matings between impotent strains that are needed to examine, for
example, whether allelism or linkage exists between various i m p mutants. The
unsuspected presence of non-selective lethal gene( s ) in these i m p strains further
complicates their analysis. Therefore, this report represents the extent to which
we feel i t is fruitful to analyze stxains imp-1 through imp-8, and we are now
screening for temperature-sensitive imp strains using a high-mating parental
strain from which lethal genes have been removed by extensive backcrossing.
General properties of impoterit mutant strains
A review of the mating process in C. reinhardi as it is presently understood
reveals that an impotent phenotype might be caused by at least four distinct
classes of mutations: Class Z mutations would prevent cells from responding to
nitrogen starvation and/or would in some other way block all gametogenic
events; CZass ZZ mutations would prevent the flagellar agglutination (clumping)
reaction that initially occurs between mt+ and mt- gametes (SAGER
and GRANICK
1954; WIESE1969; SNELL,KROOPand ROSENBAUM
1973; MCLEAN,LAURENDI
and BROWN 1974; BERGMAN
et al. 1975), analogous to a mutant strain of
Paramecium described by SONNEBORN
and LYNCH(1937) ; Class 111 mutations
would prevent the shedding of cell walls that occurs when gametes of opposite
GAMETIC D I F F E R E N T I A T I O N MUTANTS
181
mating type agglutinate (CLAES1971; GOODENOUGH
and WEISS1975); and
Class ZV mutations would prevent the formation of the cytoplasmic bridge
(FRIEDMANN,
COLWINand COLWIN1968; GOODENOUGH
and WEISS1975) and
subsequent cell fusion between m t f and mt- gametes, analogous to another
Paramecium strain, the "can't mate" mutant of SONNEBORN
(1942).
The eight impotent strains described in this report all carry out at least some
of the events accompanying gametogenesis-all, for example, acquire a mating
structure associated with their anterior cell membrane (FRIEDMANN,
COLWIN
and COLWIN3968; GOODENOUGH
and WEISS1975)-and none is therefore
considered to carry a Class I mutation. The strains imp-2, imp-5, imp-&,imp-7,
and imp-8 all fail to acquire mating-type-specific flagellar agglutinability
[although interestingly, all become agglutinable by the lectin concanavalin A
(K. BERGMAN,
unpublished observations), a property of gametic but not vegetative flagella (WIESEand SHOEMAKER
1970)], and these strains are thus of
Class 11. The rapid loss of cell walls requires mating-type-specific flagellar
and WEISS1975) and is therefore not
agglutination ( CLAES1970; GOODENOUGH
normally observed in matings involving Class I1 mutants; all of the Class I1
strains can, however, be induced to lose their cell walls by prolonged incubation
in N-free medium with mt- gametes, indicating that none carries a Class I11
mutation per se. Finally, the strains imp-I, imp-3, and imp4 carry Class IV
mutations, the latter two strains being very leaky.
A minimum of one gene locus is represented by the Class I1 imp strains
described here; the highly differing success in zygote formation exhibited by the
various strains (Table 2 ) may indicate that we have isolated different alleles of
the same locus or that certain strains (e.g., imp-7) carry additional mutations
that interfere with mating success. Two loci are represented by the mutations
zmp-3 and imp-4, and one locus is represented by imp-2. Thus our analysis
reveals that a minimum of four loci are concerned with gametic differentiation
in C. reifihardi.
Two distinct aspects of gametic differentiation can be recognized in C .
reinhardi, namely, those restricted to one or the other mating type (matingfype-specific traits) and those that are common to gametes of both mating types
(general gametic traits). Each of these will be discussed in turn and related to
the impotent strains reported in this paper.
Mating-type-specific traits
An enumeration of mating-type-specific traits includes the following: First,
the surfaces of m t f gametic flagella clearly carry one or perhaps several
agglutination factors that are distinct from, and agglutinable with, factors present
1973;
on mt- gametic flagella (WIESE1969; SNELL,KROOPand ROSENBAUM
MCLEAN,LAURENDI
and BROWN1974; BERGMAN et al. 1975). Secondly, the
mating structure of a plus gamete which mediates cell fusion is quite different in
morphology and in function from the mating structure in a minus gamete
(FRIEDMANN,
COLWINand COLWIN1968; GOODENOUGH
and WEISS1975). And
thirdly, the m t f gamete uniquely transmits to the zygote a group of genes
182
U. GOODENOUGH, C. H W A N G A N D H. MARTIN
concerned chiefly with sensitivity to antibiotics and believed to reside in
chloroplast and/or mitochondrial DNA (reviewed by GILLHAM1974 and by
SAGER
1972).
If even two mating-type-specific traits are granted, then an apparent dilemma
arises. The mating-type locus behaves in crosses as a single pair of alleles, mt+
and mt- (SAGER1955). Indeed, since C. reinhardi is a haploid organism, control
of m a t i q type must reside at a single locus since if other independent loci were to
influence mating type, then as a result of recombination, progeny would be
expected to arise in which mating type was not clearly expressed. The dilemma,
therefore, is to explain how a pair of apparent alleles can each specify more than
one mating-type-specific trait.
The simplest model to account for these observations is to propose that the mt+
locus is in fact a region of chromosome VZ which contains all the genes concerned
with the mating type plus phenotype and that recombination is disallowed in this
region of the chromosome so that these genes are always transmitted together;
similarly, the mt- locus can be visualized as a comparable region containing all
the genes concerned with the minus phenotype in tight linkage. More complex
models involving regulatory genes can, of course, also be constructed, but until
evidence for such genes is put forward, the simple model suffices to explain most
of the observations made to date.
One of these observations, first put forward by GILLHAM( 1969), is that little,
if any, recombination is in fact observed in the sector of linkage group VI
contiguous to mt. Thus at least six gene loci-ac-29, nic-7, thi-IO, cyt-1, mat-I,
and mat-2-a11 fail to recombine with mt or, where such crosses have been
performed, with one another (VVARR 1968; GILLHAM
1969; SAGER
and RAMANIS
1974). The mai-l and nzat-2 mutations affect the mating-type-specific transmission of organelle genes and thus might be expected to be closely linked to mt,
but the remaining four loci are concerned with traits that are apparently
unrelated to mating-type specification, suggesting that the postulated suppression
of crossing over in the mt region is extended to include adjacent portions of the
chromosome.
The second observation relevant to this model is that the imp-I mutation
reported here fails to recombine with mt. Gametes carrying the imp-l mutation
retain one mating type plus-specific trait (the ability to agglutinate with mtgametic flagella) but are impaired in a second mating type plus-specific trait
COLWIN
[the elaboration of a fertilization tubule during mating ( FRIEDMANN,
and COLWIN1968; GOODENOUGH
and WEISS1975) 1. It therefore seems unlikely
that imp-2 represents a deletion of the entire mt+ locus; more likely, the imp-l
mutation marks one of the several structural genes postulated to lie within the
locus.
The imp-l mutation has been shown (GOODENOUGH
and WEISS1975) to
produce structural defects in the mtf mating structure associated with the
anterior cell membrane of the gamete. Specifically, the doublet zone, which
normally generate microfilaments during the mating reaction, has a mutant
morphology in imp-I gametes and cannot give rise to filaments. Therefore, it
GAMETIC DIFFERENTIATION MUTANTS
183
ceems reasonable to infer that the imp-l mutation actually lies within the mt+
locus and affects its ability to specify a functional doublet zone. Until some
mechanism can be found to eliminate the apparent suppression of recombination
involving mt, however, tight linkage to mt cannot be cited as demonstrating that
a mutation actually lies within the locus, and a detailed fine-structural analysis
of the region cannot be performed.
General gametic traits
Turning now to the general gametic traits that are common to both mating
types, several can be listed. First, mt+ and mt- cells both appear to require the
same stimulus and the same length of time to differentiate into gametes.
Secondly, both types of gametes undergo similar structural and metabolic alteratims during gametogenesis: both, for example, degrade their ribosomes during
gametogenesis, accumulate starch, reduce their photosynthetic capacity, and
acquire a novel kind of cytoplasmic vesicle (MARTIN
and GOODENOUGH
1975).
‘Thirdly, both have flagella that are longer (RANDAIJ,et al. 1967) and apparently
more rapidly beating than vegetative flagella; both flagella types exhibit the
same sensitivities to enzymatic digestion (WIESEand METZ1969; WIESEand
HAYWARD
1972). Finally, both gamete t y p x produce a lytic activity that
mediates the shedding of cell walls during mating (CLAES1971; GOODENOUGH
and WEISS1975).
The imp-2. imp-3, imp-4, imp-6, imp-8, and probably the imp-5 mutations
described in this report are cither loosely linked or unlinked to mt, and the imp-3
and ima-4 mutations are capable of expressing their defects in either mating
type. These mutations may therefore affect genes that control general gametic
traits. It is also possible, however, that the mutations affect genes which play no
direct role in gametic differentiation. For example, one gene marked by an imp
mutation and unlinked to mating type may prove to be expressed only during
differentiation, either in direct response to nitrogen starvation or in response to
some “positive control” signal ( ENGELSBERG,
SQUIRES
and MERONK
1969) from
the mt locus, and may confer some general gametic trait on the differentiating
cells. On the other hand, a second imp mutation might perhaps affect the lipid
composition of the flagellar membrane in a way that had no effect on vegetative
cells but prevented the establishment of the agglutinable state in gametic flagella
of either mating type. The gene marked by such a mutation would not be of direct
relevance to the goal of understanding the genetic control of differentiation in
C. reinhardi.
The task of differentiating “specific” from “non-specific’’ gene mutations is, of
courze, central to developmental genetics. Ultimately, the question can only be
decided by correlating gene mutations with alterations in gene products that are
known to appear only in response to a differentiation stimulus (e.g., nitrogen
starvation). The relatively simple and increasingly well-defined phenotype of
the C. reinhardi gamete will hopefully simplify such a task.
184
U. GOODENOUGH, C. H W A N G A N D H. M A R T I N
CINDYKEESON,SAI-KITLAW,and JACKJAWITZ
participated in early phases of this research.
Conversations with PROFESSOR
R. P. LEVINEwere invaluable throughout. Supported by Grant
G M 18824 from the N.I.H. and by a grant from the Maria Moors Cabot Foundation for Botanical
Research, Harvard University.
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Corresponding editor: S . L. ALLEN