J.

Mutation induction, isolation and characterization in

Aspergillus niger.
Group #: 50
Names: Marleen van der Wiel and Mats Ewals

Mutations:
- Are the ultimate source of evolution
- Are required for gnetic analysis (markers needed)
- Arise spontaneously at low frequency but
o Can be induced (e.g. X-ray, UV, EMS)
o Can be selected (if there is an associated phenotype)
- Most but not all deleterious
- Most are recessive
 Genetic dissection of e.g. NO3- metabolism pathway.

Lifecycle A. niger

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Most mitotic divisions occur during asexual sporulation

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day 2 First watch the instruction movie.
- Count the colonies on the CMT and SMClO3 plates and fill in the Table below. Make photographs of
the plates for the report.

Conidia colonies on 0.05mL 100 0.2mL 100 0.1mL 10-4 cfu/mL in % survival % resistant
plate 100 of UV colonies
1 2
non-radiated SMCLO3 3 15 0.001
CMT 16 11 1350000
15 sec UV- SMCLO3 63 1260 0.087
irradiated CMT 20 9 1450000 107%
50 sec UV- SMCLO3 93 1860 0.311
irradiated CMT 7 5 600000 44,4%

1. Calculate the corresponding colony forming units (cfu/mL) in the original undiluted suspensions and fill
in the Table.
2. Calculate the effect of UV on survival of spores by comparing the viable count (cfu) of irradiated and
non-irradiated spores. Explain the results.
Under the influence of UV the number of colonies will be reduced due to damage to spores and
cells. However when the plates received 15 sec. of UV irradation more colonies were found,
according to the lab assistance this happens quite often and there is no explanation for this result
at this moment.
3. Calculate the frequency of spontaneous and UV-induced resistant mutants. Explain the results.
SMClO3 is a selection media where this fungus is not supposed to grow. UV is not only
responsible for damage to cells but can cause mutations in the DNA, the increase of colony
growth on the selection media when irradated longer with UV might be because of mutations
due to UV which lead to resistance cells to the SMCL0 3 media.

- Watch instruction movie. Mark the 22 colony positions on a SMClO3 plate with a plastic matrix
corresponding to the needles on the 22 needle-replicator we will later use to transfer all colonies at
once to test plates. Transfer 20 ClO3- resistant colonies by inoculating spores with the hook needle (if
there are no or few spores, use the hook needle for transfer of some hyphae) onto the marked SMClO3
plate. Indicate on the plate whether the resistant colonies are from UV-irradiated or non-irradiated
spores. Hand in for incubation at 330C for 2-3 days. We used the 50 seconds UV treatment for
transfering the colonies.
- Dispose of all counting plates in the biological waste bin (put them in carefully).

day 3 First watch the instruction movie

- Make replicates using the needle stamp onto the SM test plates with NO 3-, NO2-, hypoxanthine and
urea as N-source. Inoculate by hand on each test plate the original ancestral strain N522 for internal
reference. Hand in for incubation for 2 days at 33°C. Hand in the Master Plates for storage in the
refrigerator for later use (see day 4).

day 4 First watch the instruction movie

4. Score the growth of all 20 mutants on the different N-sources in the table and classify the mutants (see
Table 1 for classification).

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 NO3- NO2- Hypoxanthine Urea Mutant gene
N522 + + + +
1 - + + + niaD
2 - + + + niaD
3 - - + + nirA
4 - - + -
5 - + + + niaD
6 - + + + niaD
7 - + + + niaD
8 - - + -
9 - - + -
10 - - - - Not correctly
inoculated
11 - + + + niaD
12 - + + + niaD
13 - + + + niaD
14 - + + -
15 - + + + niaD
16 - + + + niaD
17 - - + + nirA
18 - + + + niaD
19 - + + + niaD
20 - + + + niaD
At some spots the inoculation did not go so well so there we scored a -
a. Check the master plate to see which colonies were present. Of those, only consider colonies that
grow on the urea plate (why?).
b. Use the growth of N522 as a reference to discriminate between wild-type (+) and mutant growth (-).
c. Make photographs of the plates for the report.
d. How many niaD, nirA and cnx(A-E) mutations did you find? Did you find a crn mutant?
niaD: 13
niaA: 2
cnx (A-E): 0
We did not find crn mutants

e. Sometimes a group finds many mutants without UV that are mostly of one type (e.g. all niaD). How
can this be explained (see exp L)?
Some mutations already pre-existed in the population because one gene is more prone to
mutations what explains this.

Some cultures may already have an early chlorate-resistance mutation. Chlorate resistant
colonies rom unradiated spores may therefore have the same mutation whereas from spores
that were UV-induced yield independent chlorate resistant colonies.

5. Explain the growth characteristic of cnx mutants.

They are able to grow both on the No2 and the urea plate. They produce white mycelium and
when they sporulate they turn light brown//yellowish

Good growth on urea, some growth in nitrite (better than control), non (only some thin growth) on
nitrate and hypoxanthine

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 Figure 2. Complementation test to determine whether two different cnx mutations are in the
same gene or not.

To further classify your cnx mutants We will now perform a complementation test (see Figure 2.) of
two of your cnx mutants with several cnx mutants of our laboratory stock. The complementation test is
based on heterokaryon formation. When this heterokaryon consists of nuclei with mutations in
different cnx-genes, the heterokaryon will grow on nitrate as N-source, hence it shows
complementation. If the mutations are in the same gene, the heterokaryon will not show growth on
nitrate.
- Inoculate on the plate SM+NO3 spores of one of your cnx mutants on places 1, 2, 3, 4, 5, 6 (see figure)
and the different cnx stock mutants on places 4, 7 and 5, 8 and 6, 9 as follows. Take a sterile
toothpick, dip in the SM plate to make it wet and take a visible sample of spores (we need quite some
spores for this test, since germinating spores are more prone to fuse and form heterokaryon than
growing hyphae) to inoculate and then put the toothpick in a Petri-dish with alcohol. Take a new
sterile toothpick for each colony. At the places 4, 5, 6 your mutant and a reference strain are
inoculated at the same spot. If the mutant and the reference strain complement each other, growth at
the “mixture spot” should be clearly different from growth at the spots of “pure strains”. Repeat this
for the second cnx mutant. Hand in for incubation at 33 oC for ~7 days. Please remove the markings
from the master plate with 70% ethanol, before you return it at the window tables.
After you are done with the experiment, return the glass master plate to us on the window side table
and carefully dispose the test plates in the biohazard bin.

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 N698 (cnxA2)
your mystery cnx
mutant
1 4 7

N699 (cnxB3)
2 5 8

3 6 9
N701(cnxC5)
)

day 5
- Score the complementation test and take a photograph. Check whether inocula 1, 2, 3 and 7, 8, 9
indeed show poor growth. Next check which of the inocula 4, 5, 6 show strong heterokaryotic growth
and which not? Check complementation of spore colour markers under the microscope.

- 6. Explain why only heterokaryons can grow on SM+NO3 in the complementation test.
Because heterokaryons contain all the genes that are necessary to grow on the SM+NO3 plate.

Both strains have a cnx mutation thus have no functional cofactor for nitrate reductase activity.
Heterokaryon of with nuclei carrying either of these recessive mutation can grow due to
complementation by the dominant wild type allele.

- 7. How can you be sure that you have indeed a heterokaryon? Hint: look at the colour of the spores, in
A. niger the colour markers fwnA1 and olvA1 show complementation too in heterokaryotic sporeheads.
When we have a heterokaryon the genes fwnA1 and olvA1 will show complementation which
will result in black spores, this is only possible when in a heterokaryon (or when two parents
were crossed) and this way we can be sure that we have indeed a heterokaryon.

Both strains have also a spore colour mutation, will also show complementation of these in
heterokaryotic conidiosphores (black spore) hence heterokaryons have olive-green, fawn and black
conidial heads.

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- 8. To which known cnx-gene is your mutant allelic?
CnxA2, because this heterokaryon was not able to grow on the SM+NO 3 media so this
heterokaryon is “homozygous (for lack of a better word)” for this mutant and can not grow.
- 9. Do you think the chlorate-resistance effect of your cnx mutant is dominant or recessive in this
heterokaryon? How would you test this?
Recessive, we can test this by replating the heterokaryons on a SM with chlorate, when this
effect is recessive, the heterokaryons are not able to grow. This is because cnxA1 will have a
mutant for cnxA1 but will have the wild type for cnxB, and the other way around for the spores
which contain a cnxA+ and a cnxB1. When these are combined the cnxA+ will be dominant over
the cnxA1 and the heterokaryon will not be able to grow on the chlorate.

Most likely recessive because in a heterokaryon of different cnx-mutants the capacity to grown
nitrate as N-source was restored. Since functional nitrate reductase gives chlorate toxicity such
heterokaryons are likely sensitive to chlorate. Could be tested in a heterokaryon or diploid.
- 10. Explain why chlorate resistance is an example of genetic heterogeneity.
In the pathway, you have several genes that affect the reduction of nitrate/chlorate and defects
in all these genes will affect the pathway

More than one gene can mutate in yield chlorate resistance.

- 11. Explain how you can select, from a mixture of spores, those with the cnx mutant allele as well as
those with the cnx wild-type allele (i.e. ‘two-way-selectable’) by using their pleiotropic effect. E.g.
how would you select for wild-type revertants from a cnx mutant strain?
We know that the cnx mutant strain is able to grow on chlorate. We can select the wild-type
revertants be plating spores of the cnx mutant strain on a plate which contains chlorate and on a
masterplate. When certain colonies are able to grow on the masterplate but not on the plate
containing chlorate, you can conclude that this is a wild-type revertant because otherwise it
would have been able to grow on chlorate.

Form a suspension of wild type spores in which there is a single cnx mutant can be selected based
on chlorate resistance. From a cnx mutant spores a single revertant spore can be selected based on
ability to grow on nitrate.

- 12. Wild-type A. niger has a typical black conidiospore colour (hence its name). Would you expect
such conidia to have a higher resistance to UV than the fawn-coloured fwnA1 mutant spores? Give a
stepwise protocol to test that.
Black spores (just like coloured humans) have a higher resistance to UV. This can be tested by
running the experiment again by including both the black conidiospores and the fawn-coloured
mutant spores and counting the survival of both the black conidiospores and the fawn coloured
ones.

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