Pale Chlamydomonas in TAP in semi-dark?

Elizabeth Harris chlamy at ACPUB.DUKE.EDU
Thu Aug 31 16:48:01 EST 1995


This is a question that arises frequently, so I'm posting my reply to the
newsgroup for the benefit of others.

Nicholas Bertos wrote,

>I've got a question about what Chlamy (UTEX 90, a wild-type) is supposed to
>look like when it is grown in Tris-acetate-phosphate medium (recipe from
>Chlamy handbook) at about 250 lux/20 foot-candles. Mine are rather diffcult
>to grow; growth is very slow and the cells appear yellowish-pale green
>rather than the usual darker green. When looking at these cells under the
>electron microscope, the thylakoids were extremely reduced and there were
>starch granules all over the place.
>
>According to Dr. Wollman, my Chlamys are mutated/drifted from wt to a
>phenotype resembling y-1 light-only greening mutants. Since I've gotten the
>same result with two batches that I ordered from UTEX, I'm not sure if this
>is what happened, or maybe if the original stock culture at UTEX may have
>drifted. Is there any other possibility for the strange appearance of these
>Chlamys? Thanks in advance for any help!

You're almost certainly looking at the y-1 mutation, and we see the same
thing in our stock corresponding to UTEX 90:

CC-1010 wild type mt+ (UTEX 90)
=46rom UTEX collection 5/80
Note: UTEX identifies as mt-, but tests by us show mating with CC-124.
Originally from G.M. Smith, given by J.N. Hartshorne to Culture Collection
of Algae and Protozoa, Cambridge (=3D their 11/32a).  Stock from Cambridge
sent to Indiana (now UTEX) collection, UTEX stock sent to SAG collection in
G=F6ttingen, where it became their 11-32b.
May or may not be from same Smith isolate as Levine and Sager wild type
strains.  Chloroplast DNA is indistinguishable from Levine wild type with
Bam HI, Bgl I
This stock grows on nitrate.
Both Richard Starr and Graham Bell report problems with this stock growing
in dark 1991

When I tested this stock myself, I found that it had a high proportion of
y-1 cells.  The light intensity you describe is probably not sufficient to
override the y-1 phenotype.

There are two possible remedies:

1) Get a different wild type strain.  We would be happy to send you a wild
type that it is green in the dark.

2) Subclone the one you have.  The y-1 mutation has a high interconversion
rate with its wild type allele (reported to be as much as 1 in 10^3 in some
strains), but mayy has some selective advantage in strains grown in
continuous light, since it tends to accumulate in many cultures.  Still,
with this kind of frequency, you have a very good chance of picking up a
green colony.  Make a dilution of your stock such that 0.1 ml spread on a
petri plate will give about 100-200 colonies.  Make several spread plates,
and wrap them in foil.  Look at them after a week to 10 days, and try to
find a colony that looks greener than most.  Pick that off, streak it out
on a fresh plate, grow it up, and repeat the subcloning procedure.  (It's
going to take 2 rounds minimum to get a pure green clone.)


Elizabeth Harris
chlamy at acpub.duke.edu






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