Subject: FlyBase query
Dear Dr. Kalderon,
I am curating the following paper for FlyBase:
Davis et al., 1998, Neuron 20(2): 305--315
(Postsynaptic PKA controls quantal size and reveals a retrograde signal
that regulates presynaptic transmitter release in Drosophila).
in which they use two constructs containing 'a mutant PKA regulatory
subunit fused to the UAS promoter' which they obtained from you (they also
reference Li et al., 1995, Cell 80: 553--562 for these constructs).
The two constructs are: (<up></up> = superscript)
1) UAS-PKAinh1 (stated to formerly be UAS-PKABDK22) which carries a
single mutation in a cAMP binding site.
2) UAS-PKAinh2 (stated to formerly be UAS-PKABGD) which carries mutations
in two cAMP binding sites.
a) Could you confirm that these are constructs using the Pka-R1 gene rather
than the Pka-R2 gene.
b) FlyBase has a record of a single UAS-PKA regulatory subunit construct
from Li et al., 1995, Cell 80: 553--562. This is called UAS-R* in that
paper. I would be grateful if you could tell me whether either
UAS-PKAinh1 or UAS-PKAinh2 correspond to UAS-R*, or whether they are 3
separate constructs.
I look forward to hearing from you,
Gillian Millburn
\--------------------------------------------------------------
Gillian Millburn.
FlyBase (Cambridge),
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>
Subject: Re: FlyBase query
Dear Gillian,
	All of the constructs used RI. The UAS-R* of Li et al really
refers to both constructs as both gave the same results in these exp'ts
although BDK (as opposed to BGD) was used much more frequently (and for
example is used exclusively in our latest paper, Ohlmeyer et al., 1997
in Genes & Dev). From reading your summary I realize that there must
also be a mistake in the Davis paper since BDK has two mutations and BGD
only one. This is probably not very important since both constructs work
as inhibitors; mutation of both sites slightly reduces affinity for
catalytic subunit in vitro and makes R entirely insensitive to cAMP- the
single site mutant can be activated in vitro but by cAMP concentrations
higher than those likely to be seen in vivo. Probably none of these
details will ever be published so I realize that the UAS-R* will remain
incompletely documented in the literature.
Sincerely,
Daniel Kalderon
>
Subject: Re: FlyBase query
Dear Daniel,
thankyou for your prompt reply, it was very helpful. I have a few more
questions to sort out the details of the two UAS-Pka-R1 constructs, and
then if it is OK with you I would like to include the information about the
two constructs in FlyBase as a personal communication from you.
1.
I would like to confirm that:
a) the constuct with a mutation in 1 cAMP site is called BGD
b) the construct with mutations in 2 cAMP sites is called BDK
2.
Would you prefer the constructs to be called:
P{UAS-Pka-R1.BGD} and P{UAS-Pka-R1.BDK}
or, as used in the Davis paper:
P{UAS-Pka-R1.inh1} - for the construct with a mutation in 1 cAMP site
P{UAS-Pka-R1.inh2} - for the construct with mutations in 2 cAMP sites
I look forward to hearing from you,
Gillian Millburn
>
Subject: Re: FlyBase query
On Thu, 12 Mar 1998, Gillian Millburn wrote:
> Dear Daniel,
>
> thankyou for your prompt reply, it was very helpful. I have a few more
> questions to sort out the details of the two UAS-Pka-R1 constructs, and
> then if it is OK with you I would like to include the information about the
> two constructs in FlyBase as a personal communication from you.
>
> 1.
> I would like to confirm that:
>
> a) the constuct with a mutation in 1 cAMP site is called BGD
> b) the construct with mutations in 2 cAMP sites is called BDK
That is correct. The BGD mutation is in the second cAMP binding domain
(more C-terminal) changing G321 to D. BDK additionally has a mutation
G196 to E in the analogous position in the first cAMP binding domain>
> 2.
> Would you prefer the constructs to be called:
>
> P{UAS-Pka-R1.BGD} and P{UAS-Pka-R1.BDK}
> Above is better because the Davis paper is a potential source of
confusion and when I send out stocks they always say BDK or BGD
> or, as used in the Davis paper:
>
> P{UAS-Pka-R1.inh1} - for the construct with a mutation in 1 cAMP site
> P{UAS-Pka-R1.inh2} - for the construct with mutations in 2 cAMP sites
>