Subject: RE: Help FlyBase please
P element transformation experiments with Orct failed to rescue the lmg
phenotype and we subsequently discovered an adjacent gene, containing the
l(2)03424 P element insertion in its 5' untranslated region.
The current state of affairs is summarised in the abstract of the poster at the
Zurich meeting, a slightly updated version of which I attach below:
Mutations in the lemming (lmg) gene lead to abnormal mitoses and apoptosis in
imaginal cells. Over-condensed chromosomes and polyploid nuclei are observed
in larval neuroblasts. Lagging chromosomes are observed during anaphase in
abdominal histoblasts undergoing their second mitosis following pupariation.
These cells appear to exit from mitosis into a G1-like binucleate state or
undergo apoptosis directly from mitotic arrest. Widespread apoptosis is also
observed in imaginal discs of lmg mutant larvae.
The lmg gene was cloned by virtue of a P element insertion in the 5'
untranslated region (l(2)03424). lmg mRNA is found uniformly in early embryos,
becoming restricted to the central nervous system in older embryos, and
imaginal discs in larvae. The gene is also expressed in ovarian nurse cells in
adult females. Alternative transcripts are observed at different developmental
stages. The predicted protein coded by the lmg gene is homologous to the
APC11p component of the anaphase promoting complex (APC) of Saccharomyces
cerevisae and has homologues in humans and the nematode Caenorhabditis elegans.
These proteins belong to a larger family of small, highly conserved RING zinc
finger-containing proteins.
Best wishes,
Alan
>
Subject: RE: Help FlyBase please - Orct and lemming
Dear Dr. Shirras,
I am a curator working for FlyBase. Michael has passed on to me your
e-mail concerning Orct and lmg, so that I can record the information in
your e-mail as a personal communication from you to FlyBase, and so
that I can correct the information in the database.
I have some questions which arise from Figure 1. in your paper:
Taylor et al., 1997, Gene 201(1-2): 69--74
At the moment lmg and Orct are in FlyBase as one gene and I want to
make sure that I get the accession numbers and functions etc. with the
right genes when I make the corrections in the database to make them 2
separate genes.
1. Presumably Acer (which is in Fig. 1 of the Gene paper) is not the
lmg gene, as in your e-mail to Michael you state that the l(2)03424
insertion which causes the lmg phenotype is in the 5' untranslated
region of the lmg gene ?
2. In Figure 1. of the Gene paper, you show 2 cDNAs - R3 (accession
number Y12399) and 7d (accession number Y12400) which are Orct cDNAs.
I would just like you to confirm that these are still thought to be
Orct cDNAs and not lmg ones. Also do you still think that the 5' ends
of these cDNAs map upstream of Acer, so that it is within an intron of
Orct (in which case lmg must also be in an intron of Orct)?
I look forward to hearing from you,
Gillian
\--------------------------------------------------------------
Gillian Millburn.
FlyBase (Cambridge),
\--------------------------------------------------------------
>
Subject: RE: Help FlyBase please - Orct and lemming
> I have some questions which arise from Figure 1. in your paper:
Dear Gillian,
> 1. Presumably Acer (which is in Fig. 1 of the Gene paper) is not the
> lmg gene, as in your e-mail to Michael you state that the l(2)03424
> insertion which causes the lmg phenotype is in the 5' untranslated
> region of the lmg gene ?
>
- Correct.
> 2. In Figure 1. of the Gene paper, you show 2 cDNAs - R3 (accession
> number Y12399) and 7d (accession number Y12400) which are Orct cDNAs.
>
> I would just like you to confirm that these are still thought to be
> Orct cDNAs and not lmg ones. Also do you still think that the 5' ends
> of these cDNAs map upstream of Acer, so that it is within an intron of
> Orct (in which case lmg must also be in an intron of Orct)?
>
Yes, R3 and 7d are still thought to be Orct cDNAs and the 5' ends map
upstream of Acer. Both Acer and lmg lie in an intron of the R3 form of Orct,
lmg being transcribed in the opposite direction. Thanks to the genome
sequencing projects, we have found that the structure of the 7d transcript
shown in the paper is wrong - what we have shown as the downstream exon of 7d
is in fact another organic cation transporter gene.
Hope this helps.
Alan
>
Subject: RE: Help FlyBase please - Orct and lemming
Dear Alan,
thanks for your reply, it was very helpful. I have one more question:
you wrote:
>Thanks to the genome
>sequencing projects, we have found that the structure of the 7d transcript
>shown in the paper is wrong - what we have shown as the downstream exon of 7d
>is in fact another organic cation transporter gene.
do you have a name for this second organic cation transporter gene,
e.g. Orct2, so that I can include the information that this is a
separate gene. Also, this presuambly means that the accession numbers
Y12400 (Genbank) and O01384 (SwissProt) contain sequences belonging to
both Orct and the second organic cation transporter gene.
Gillian
>
Subject: RE: Help FlyBase please - Orct and lemming
Dear Gillian,
> do you have a name for this second organic cation transporter gene,
> e.g. Orct2, so that I can include the information that this is a
> separate gene.
>
No, we have not characterised this gene and have not given it a name,
though Orct2 would be appropriate.
> Also, this presuambly means that the accession numbers
> Y12400 (Genbank) and O01384 (SwissProt) contain sequences belonging to
> both Orct and the second organic cation transporter gene.
>
No, it was not a hybrid cDNA so the sequences are fine. The confusion
arose because of cross-hybridisation on Southerns.
While you're about it, you may wish to know that there is a gene, which
we have called anon-29Da, situated between the downstream Orct R3 exon and
Orct2 and transcribed in the same direction as Orct. We have submitted two
sequences of alternative transcripts, accession numbers AJ249493 and AJ249494.
Alan