comai at U.WASHINGTON.EDU
Sat Apr 24 17:21:17 EST 1993
below are several answers to my query on genes used as loading controls in
northern blots. I have added some references pulled from AATDB. I wish to
thank all those who provided information.
Cytosolic cyclophilin (CyP). Gasser's lab (csgasser at ucdavis.edu)
Cytosolic cyclophilin genes appear to be expressed in
all cells of all organisms examined from all kingdoms. In
our published (Gasser et al., 1989, Proc. Natl. Acad. Sci.
87:9519-9523) and unpublished work we have found very
consistent high levels of expression in all tested organs and
tissues of higher plants (roots, leaves, fruits at various
stages, stamens at various stages, pistils at various stages,
the shoot apex, sepals, petals). We have recently cloned the
Arabidopsis cytosolic CyP gene and find that it gives a
signal that is easily detectable in eight hours on northern
blots with 20 ug of total RNA. As seen for the other plants,
the levels of RNA are constant in roots, leaves, and flowers.
We routinely use the CyP probe as a control for the amount of
RNA on our northern blots with samples from different organs.
Cytosolic CyP has been reported to be induced several fold by
heavy metal treatment or wounding (Marivet, et al.,1992,
Plant Sci. 84(2): 171-178.) but it does not seem to be
induced by ethylene (our data). Thus, CyP may not be a good
control for stress experiments. The Arabidopsis cytosolic
CyP we have isolated is different from another published CyP
from Arabidopsis (Bartling,et al.,1992. Plant Mol. Biol.
19(3): 529-530.) for which the expression pattern has not
been reported. We are glad to send a sample of a plasmid
which can be used as a probe to any interested laboratory.
When our publication on this gene comes out we will deposit
the clone at the Ohio State stock center for distribution.
CSGASSER at UCDAVIS.EDU
5S and 18S rRNA genes (Zimmerman's lab)
We are using 5S or/and 18S rRNA genes as controls in our (Dr. Lynn
Zimmerman's) lab, assuming they are constitutively expressed and take the
majority (>90%) of the transcripts. The 5S gene is from soybean, the 18S
gene is from Xenopus,
they work fine with carrot.
* Biological Sciences Internet: xlin at umbc.edu *
* U. of Maryland Baltimore County Lab Phone: (410) 455-2263 *
* 5401 Wilkins Avenue Fax: (410)-455-3875 *
* Baltimore, MD21228 *
* Home Phone? Can't be fingered out. I am using my wife's account. *
Tubulin (Poethig's lab)
I have used tubulin genes as controls in RNase protection experiments. I
got the clones from 2 different sources - from David Marks (Lincoln,
Nebraska), and Patrick Hussey (Queen Mary and Westfield college, University
of London, UK). Let me know if you want more detailed info - or maybe
you'll hear from them. I found the mRNA I detected with these probes was
not very very abundant, so if quantity is a problem you might want to think
about something else. I think you also need to consider what you are
controlling for - are your genes expressed in leaves, in response to light,
stress etc? Because some of the apparently "constitutive" genes may also
be affected by certain stimuli. The other genes we considered are rubisco,
actin, chalcone synthase etc,
Yours, Emily Lawson (in S.Poethig's lab)
spoethig at mail.sas.upenn.edu
Snustad, D. P., Haas, N. A., Kopczak, S. D., Silflow, C. D.
The small genome of Arabidopsis contains at least nine expressed
beta-tubulin genes. The Plant Cell, 1992, 4: 549 556
(Department of Genetics and Cell Biology, University of Minnesota, St.
Kopczak, S. D., Haas, N. A., Hussey, P. J., Silflow, C. D., Snustad, D.
P. The small genome of Arabidopsis contains at least six expressed
alpha-tubulin genes. The Plant Cell. 1992. 4. 539-547
actin (Federoff's lab) -it was probably an heterologous actin
Nairn, C. J., Winesett, L., Ferl, R. J. Nucleotide Sequence of an Actin
Gene from Arabidopsis thaliana Gene (Amsterdam) 1988. 65:247-258
Ubiquitin (Meyerowitz's lab)
We have used a ubiquitin (common for UBQ5 and 6) probe successfully for
Northerns. I amplified a short fragment from genomic DNA, corresponding
334-585 of UBQ5. Reference: Callis et al., JBC 265, 12486 (1990).
WeigelD at starbase1.caltech.edu
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