Subject: Helping FlyBase: ADRC-10455
Dear Heidi,
We are currently curating the abstracts for the upcoming 42nd
(Washington, D.C.) Annual Drosophila Research Conference, for FlyBase.
I am writing in connection with your abstract:
'Gene structure and developmental regulation of the small conductance
calcium-activated potassium channel gene (dSK) in Drosophila.'
You mention a small conductance calcium-activated potassium channel gene, dSK.
Do you know which of the Genome Project CG annotations your gene
corresponds to? All the CGs have corresponding gene records in
FlyBase already and we don't like to make duplicate records for what
is actually the same gene unless we can't avoid it. If your gene does
not correspond to a CG then perhaps you could tell me its map location,
as this is valuable information for the genome annotation project.
I am writing to you because the first author does not have an address
in FlyBase \- please feel free to pass my query on if there is someone better
placed to answer it.
Thank you for your help,
with best wishes,
Rachel.
Subject: Re: Helping FlyBase: ADRC-10455
Rachel Drysdale,
Sorry for the delay but I had to check the latest and this is a
crushing week befor an overseas trip. So here is a detailed answer to
your question and a background for our efforts and plans-the later are
not fixed and we are open to suggestion.
Rational: A few years ago, I was involved in some exploratory projects
concerned with the detection, cloning and sequencing of transcripts of
potential homologues of the rat Ca-activated K-channels (rsk 1-3) in
other vertebrates (ferret, rabbit) and several invertebrates, including
mollusks and Drosophila. Several different degenerate primer pairs
produced PCR bands of the expected sizes in virtually all of the
tissues explored, including Drosophila. All the ?expected? PCR bands
were cloned and sequenced with the exception of the Drosophila PCR
bands (due to insufficient time, resources and interest among my
colleagues who worked with Drosophila). With the teaching
responsibilities for an 'Introduction to Bioinformatics', the timing of
the release of 'dripping wet' Drosophila genomic sequence and the
potential interest of a new faculty member, Heidi Theisen, it seemed a
propitious time to teach the students about the art of gene-finding and
to present them with an exciting and timely opportunity to walk a
'virgin path'. Thus, I assigned them a project that I had some reason
to believe would both challenge and reward them, by giving them the
assignment to find the homologue(s) of the rsk (1-3) in the recently
released Drosophila genome.
We began in March of 2000 by using portions of rsk2 nucleotide sequence
to search 'fresh' Drosophila genomic sequence through Berkley Flyblast
and retrieving the sequences of these two unordered sequences.
AC014402 :  CSC:AC014402  . Working draft; 29275 bases
AC014401 :  CSC:AC014401  . Working draft; 11790 bases
These clones were translate in entirety in 6-frames and peptide
segments similar to those published for rat Ca-activated K-Channel
(rsk2) were identified among the frames 4-6 of translation (primary
effort of Philip Nguyen, grad student in Chemistry). These
bioinformatics studies permitted us to order and link (or join) these
two sequencing fragments when Celera had not. It is possible that what
mislead Celera and their gene-finding algorithms was the sequence we
observed of a transposable element found on the lower strand within the
large intron that resides between the two alternatives for exon-6. Our
'manual' gene-finding exercises produced a hypothetical (12 exon)
sequence (with alternatives for exon-6) that we believe encodes the
full length of Drosophila homologues of the rsk genes. In the fall of
2000, wet-lab experiments in Heidi's lab (Gaby Nolazco grad student)
involving RT-PCR of RNA extracted from Drosophila larvae, provided
proof (through cloning and sequencing) that both of these predicted
alternative transcripts are indeed expressed and Gaby will report on
these findings at the Drosophila meeting. ...
Now, in answer to your query, the most recent information made
available on the web suggests that our transcripts actually span two
separate predictions of Celera:
gb|AAF46030.1| (AE003434) CG10706 gene product <up>Drosophila melanogaster</up> and
gb|AAF46029.1| (AE003434) CG4179 gene product <up>Drosophila melanogaster</up>
both of which are now ordered, contiguous with each other and in the
upper strand on the X chromosome, scaffold AE003434
(spanning a 40kb region roughly from position's 40,000-80,000).
regards
Richard E. McCaman, Ph.D.