Subject: Expression in Neurospora

From: [email-masked] (Dan Ebbole)
Date: 26 May 1995 11:22:16 -0700

Geoff Turner
 University of Sheffield  found:

We wish to overexpress N. crassa glutamate dehydrogenase, 
since we are working on its structure, and E. coli is 
found to give insoluble, inactive protein.
We turned to N. crassa, and used the grg-1 expression vector 
deposited at the FGSC.  Our initial transformants with 
grg-1 driving the am gene (=gdh) are rather disappointing.

Interesting observation.  I've not noticed that subculturing leads to a
loss of expression of genes in transformed strains. Our experience has been
that different levels of expression can be obtained with SOME promoters
even when integrated at the same position in the genome.  I'm not sure how
stable this expression is during subculturing.  With the con-10 gene
expression is reproducible between independent transformants and over time
with culturing.  So it looks like there may be some Grg-1 specific problem.
 I guess these are ectopic transformants and unsure how this might affect
things.  

I would indeed consider expression in A. nidulans since the expression
systems are better worked out for Aspergillus species.   I do think that
Neurospora is a "suitable" host given some effort, but you (and I, and
everybody else) have time constraints that prevent a real effort to develop
expression systems in Neurospora when they might not be any better than
those already developed for Aspergillus.  

currently grg-1 and qa-2 are the only regulated promoters that seem to have
attracted attention for regulated overexpression of genes in Neurospora. 
------------------------
Dr. Daniel Ebbole
[email-masked]
Assistant Professor
Dept. of Plant Pathology and Microbiology
Texas A&M University
College Station, TX 77843-2132
USA

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From: [email-masked] (Dr W Dorsey Stuart)
Date: 26 May 1995 15:30:45 -0700

This may be of interest to academic labs thinking about expression in 
Neurospora.

> those already developed for Aspergillus.  

The Neugenesis corporation of Honolulu Hawaii is in fact actively 
developing expression systems in Neurospora.   We are currently 
collaborating with a major international corporation to produce a kit 
which will contain expression vectors containing both constitutive and 
regulated promoters and a cell line which has been engineered for 
overexpression of recombinant proteins,
> attracted attention for regulated overexpression of genes in Neurospora. 

Our system utilizes three regulated promoters and two constitutive 
promoters and we expect it to be available to academic labs this year.   
As we develop the kit, we will be looking for a small number of labs to 
become "alpha" testers of the system.   If you are interested in trying 
out a model version of our kit sometime within the next 60-90 days please 
let me know.

Thanks and Aloha

W. Dorsey Stuart
Neugenesis Corporation
and
Department of Genetics and Molecular Biology
School of Medicine
University of Hawaii
[email-masked]
Voice (808)-956-5515
Fax (808)-539-3804
Honolulu Hawaii 96822

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From: [email-masked] (G Turner)
Date: 30 May 1995 13:06:24 GMT

Dan Ebbole ([email-masked]) wrote:
: Geoff Turner
:  University of Sheffield  found:

: We wish to overexpress N. crassa glutamate dehydrogenase, 
: since we are working on its structure, and E. coli is 
: found to give insoluble, inactive protein.
: We turned to N. crassa, and used the grg-1 expression vector 
: deposited at the FGSC.  Our initial transformants with 
: grg-1 driving the am gene (=gdh) are rather disappointing.

: stable this expression is during subculturing.  With the con-10 gene
: expression is reproducible between independent transformants and over time
: with culturing.  So it looks like there may be some Grg-1 specific problem.
:  I guess these are ectopic transformants and unsure how this might affect
: things.  

Dan,
Thanks for the useful comments.  It might be a promoter related effect, 
but it is also true that our transformants are ectopic.
Our results are also similar to those found by Bull and Wootton,
1984, who also used gdh to transform an am- deletion strain.
However, they used only the unmodified gene fragment of gdh.
They never got up to wild-type gdh activity in their 
transformants.
All the best
Geoff

--
 Geoff Turner
 University of Sheffield

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From: [email-masked]
Date: 1 Jun 1995 10:47:22 GMT

> and Dan Ebbole replied:
> So it looks like there may be some Grg-1 specific problem.

Geoff,
in my opinion this need not to be a promotor related effect.
To constitutively express a protein in N. crassa, I tried both, 
mtr and gpdA promotor using the pBAR expression vectors deposited 
at FGSC. Both didn't work with different Neurospora genes. More 
interestingly, as a control experiment I inserted genomic DNA of 
two genes in pBARKS1 and both genes were expressed under the 
control of their own promotor. Almost all stable heterokaryotic 
transformants showed expression. 
So I can conclude that ectopic integration isn't the problem. 
A promotor related effect seems unlikely too, given the fact that 
none of the three promotors works. I'm currently trying to 
construct some new plasmids with mtr and gpdA promotors and hope
I'll get them to work.
In our lab, we had good experiences with the qa2 promotor (Fecke et al. 
FGNL 40:34, 1993) but induction with quinic acid isn't an ideal system 
for biochemical studies of purified Neurospora proteins.
I would appreciate it very much if we could discuss this problem in
more detail! 

Christoph Kruell
Universitaet Duesseldorf, Germany
[email-masked]