1. Random mutagenesis via glass-beads high-frequency transformation of Chlamydomonas reinhardtii
for fusion defective mutants
Arelis Joa Zhou and Charlene L. Forest, Department of Biology, Brooklyn College-CUNY
The focus of this study is to generate fusion-defective clones of Chlamydomonas
reinhardtii by means of radom insertional mutagenesis. To achieve nuclear transformation
of C. reinhardtii, we are using the glass-beads high-efficiency nuclear transformation
method and the sru-2-23- C. reinhardtii strain. This method involves vortexing the wall-
less C. reinhardtii cells in presence of plasmid pS103, which provides paromomycin
resistance, and acid-washed glass-beads. The use of the glass-beads aids in weakening
the cell membrane to increase the efficiency of C. reinhardtii cells to take up and hopefully
insert the plasmid into the gene responsible for fusion. Transformed cells are plated into
paromomycin plates to select for successfully transformed cells which express resistance
to the antibiotic. These cells are subject to streptomycin selection which takes advantage
of the uniparentally inherited gene for streptomycin resistance of the sru-2-23- strain. This
method will select for the transformed cells that were not able to mate or have not mated.
Once multiple fusion-defective mutants are generated, we will be able to locate and study
the genes involved in fusion of C. reinhardtii.
The conversion of vegetative cells to germ cells in C. reinhardtii is induced by nitrogen
starvation (Umen, 2011). As germ cells, when opposite mating types are mixed, four
mating stages take place: (1) agglutination, (2) activating of mating apparatus, secretion
of enzyme autolysin, followed by cell-wall digestion, (3) mating structure adhesion, and
finally (4) gamete fusion, see Figure 3. Two groups of gamete fusion-defective mutants
were created from previous studies (Figure 3).
The first group of mutants (imp-1, fus, bs-27, imp-11) is capable to proceed to stage 2 of
mating, but is defective thereon. The second group of mutants (gam-I-II, gam-10, gam-11)
consists of sex-limited mutants and are only expressed in mt-. These mutants can
proceed through mating stage 3 but the last stage, gamete fusion is blocked (Forest,
1987). HAP2/GCS1, a sperm specific gene involved in fertilization in higher species, was
also found in C. reinhardtii. Thus a mutation in this sex-limited gene in C. reinhardtii will
block its function, causing a fusion defect. The existing fusion-defective mutants (gam-I-II,
gam-10, and gam-11) plus newer insertional mutants are not defective in HAP2/GCS1.
This suggests that there may be a gene other than GCS1 involved in gamete fusion.
Therefore it is useful to generate additional fusion-defective mutants to further investigate
and determine if any other gene(s) are necessary for fusion.
Chlamydomonas reinhardtii (shown in Figure 1) is
a species of flagellated unicellular algae
commonly found in soil and fresh water
environments. This species can proliferate in two
ways, through the vegetative life cycle or the
sexual life cycle by means of mitosis or
fertilization and meiosis, respectively (Figure 2).
The ability to give rise to progeny by sexual
reproduction via gamete fusion is the focus of our
study in the lab. Like many complex eukaryotes,
C. reinhardtii have opposite mating types,
identified as mating type minus (mt-) and mating
type plus (mt+).
pSI103 isolation
The QIAGEN Midi kit protocol was followed to harvest
plasmid pSI103. The isolated plasmid, approximately
5000 bp, is shown in Figure 5. This estimated size is
close to the reported size of pSI103, 4982 bp. We are
using this plasmid to take advantage of its selective
marker, conferring resistance to paromomycin and its
high transformation efficiency due to the combined
promoters In1 and hsp-rbcS2 (Sizova et al, 2001).
Autolysin extraction
Equal amount of gametes of each mating type were mixed for 30 min. and centrifuged at
5K rpm for five minutes at 4 degrees Celsius. The crude extract was filtered using 2.7 µm
and 1.2µm filters. A final filtration (0.45 µm) and sterilization was done using the Corning
50 L tube top filter system. Approximately 35 ml of light pink autolysin was extracted.
Restriction Digestion of pSI103
The enzyme HindIII was used to cut plasmid pSI103
once. Figure 6 shows the resulting linearized plasmid.
The fourth lane (AUD) was loaded with undigested
plasmid sample while the fifth lane was loaded with
the digested sample.
Transformation of sru-2-23-
The C. reinhardtii sru-2-23- cells were transformed using Kindle’s high-frequency glass-
beads transformation method. The cells were grown for five days to a density of 6 x107
cells/ml. These cells were pelleted by centrifugation and incubated in autolysin for 45 min.
to digest the C. reinhardtii cell-walls. The wall-less cells were centrifuged at 5K rpm for 5
min. and then re-suspended using liquid TAP medium. Then plasmid pSI103 and acid-
washed glass-beads were added to the re-suspended cells. The cells, plasmid, and glass-
beads were vortexed at maximum speed for 20 seconds to allow the plasmid to enter the
cells. Thetransformed cells were then transferred to 50 ml centrifuge tuves and diluted
with 20 ml of liquid TAP medium. The diluted cells were left to recover overnight under
light at room temperature. Finally the cells were centrifuged and plated onto paromomycin
(10 µg/µl) plates. Surviving cells were subjected to streptomycin selection.
Streptomycin selection
Transformed sru-2-23- (mt-) gametes were mated with wild type R+ (mt+) gametes for
several hours. The mated cells were diluted with N-free medium. The zygotes will show
sensitivity to streptomycin, a characteristic of mt+, right after gamete fusion. The unmated
cells were separated from zygotes by filtration (10 µm), and centrifuged for 20 minutes at
7K rpm. Then cells were re-suspended with 1 ml of liquid TAP and plated onto
streptomycin (200 µg/µl) plates. The streptomycin kills all the R+ cells and zygotes. Only
unmated sru- cells will survive on streptomycin plates. Surviving transformed sru- cells are
then subjected to further screening for mating-deficient clones.
Screening for mating-deficient clones
Cells from streptomycin selection were grown for seven days and then these were
picked and plated onto solid TAP medium. After seven days, each clone was starved
for three hours in 200µl N-free liquid medium. The same amount of wt+ (R+) cells
were also nitrogen-starved for three hours. Once these are germ cells, they were
mixed and mate for several hours. Each clone was observed to determine whether it
is mating-defective.
Transformation of sru-2-23- C. reinhardtii was a success as colonies grown on
paromomycin plates indicate that the pSI103 is being expressed.
The clones collected thus far express mating-deficient due to flagella paralysis
and weak agglutination.
At this moment, a fusion-defective mutant has not been found although clones 14
and 152 will be further analyzed.
If no fusion-defected mutants are found, additional transformation experiments
will be carried out.
However, if fusion-defective mutants are found, further analysis will be carried out
using SiteFinding-PCR to determine the insertion location of pSI103 in the C.
reinhardtii genome.
1. Forest, C. 1987. Genetic control of plasma membrane adhesion and fusion in
Chlamydomonas gametes. Journal of Cell Science, 88: 613 – 621.
2. Hallmann A. 2011.Evolution of reproductive development in the volvocine algae. Figure 2.
Journal article image. Sex. Plant Reprod. 24(2): 97-112.
3. Kindle, K. L. 1990. High frequency nuclear transformation of Chlamydomonas reinhardtii.
Proc. Natl. Acad. Sci. USA. 87: 1228 – 1232.
4. Sizova et al. 2001. A Streptomyces rimosus aphVIII gene coding for a new type of
phosphotransferase provides stable antibiotic resistance to Chlamydomonas reinhardtii.
Gene. 277, 221-229
5. Umen, JG. 2011. Evolution of sex and mating loci: An expanded view from Volvacine
algae. Curr Opin Microbiol. 14(6): 634-41.
Figure 1 Diagram of Chlamydomonas
from Cronodon.com
Figure 3 Chlamydomonas reinhardtii mating stages (Forest, 1987).
Figure 5 Electrophoresis gel of isolated pSI103
Figure 6 Electrophoresis gel of
undigested pSI103 (AUD) & digested
pSI103 (AD).
Figure 4 Transformation of C. reinhardtii
Figure 7 Picked colonies from streptomycin selection.
Clone Mating Swimming Agglutination
A-004 - +/- ~
A-006 - +/- ~
A-014 - + ~
A-035 - ~ ~
A-045 - - -
A-084 - - ~
A-152 - + ~
A-166 - - -
A-192 - +/- ~
B-128 - - -
B-168 - - -
B-177 - - -
B-179 - - -
B-180 - - -
B-190 - - -
B-191 - - -
B-210 - - -
ABSTRACT METHODS & MATERIALS
CONCLUSION
REFERENCES
Screening Results
Table 1 shows the results from the
latest screening tests. The clones
shown on the table are the clones which
are mating-deficient. These mutants
were further observed under the
microscope for their swimming ability
and agglutinating ability. The (+/- )
results represent those colonies where
some cells are able to swim and some
others are not able to. The (~) results
indicate weak agglutination in
comparison to wild type agglutination.
Figure 8 C. reinhardtii strains and mated samples.
R+ NO- SRU- R+
and
NO-
R+
and
SRU-
R+
and
NO-
R+
and
A014
Figure 9 Screening samples of clone A014 and control
Figure 2
Chlamydomonas
reinhardtii life cycle
diagram from
smartsite.ucdavis.edu
animation
INTRODUCTION