>Hi Markus,
>
>I had a thought. I wonder if your PPH13 corresponds to 60Mun2, which has
>been molecularly mapped to be next to Gsc (=PPH25).
>
>Gsc corresponds to '60Mun1' published by Dessain and McGinnis
>(FBrf0065373). In this paper they discuss '60Mun1'(=Gsc/PPH25) and 60Mun2,
>on page 26. (They also say both genes are at 60C but this turned out to be
>a case of mistaken telomeres). The FlyBase gene record for 60Mun2 is at
>the bottom of this message. I can send you a copy of the relevant pages of
>the article, or all of it if you like, if you don't have a copy.
>
>60Mun2 does not have a sequence accession number but the paper gives a
>short amount of amino acid (not DNA, sadly) sequence. It is
>
>VWFK NRRAKWRKQ KREEQE
>
>and falls in the III/VI region of the homeodomain.
>
>The authors do not say that this is a prd class homeodomain, but I do not
>know whether anyone could, on the basis of such a short sequence (I am not
>a sequence expert by any stretch of the imagination, when I saw the
>transcript of my PhD gene was going on for 10kb I put my pipettes down and
>wrote up!).
>
>They do say
>'The 60Mun1 (i.e. Gsc/PPH25) partial homeobox sequence maps 4kb 3' to the
>60Mun2 homeobox, in the same 5' to 3' orientation.....'
>
>so that suggests that its going the opposite way. But I really don't know
>how carefully this was done, especially given that no-one has published on
>it since, and the cytology at least was apparently not done very
>carefully.
>
>What do you think?
>Is it possible to tell anything from such a short run of amino acid
>sequence? Does it look familiar to you? Given what you know and what
>Dessain and McGinnis say, it is possible that PPH13 could lie between Gsc
>and 60Mun2, having escaped detection, or is it more likely that PPH13
>corresponds to 60Mun2 and that the statement about the orientation of
>60Mun2 in FBrf0065373 is wrong?
>
>Hope this is not a nuisance, but if we can merge PPH13 with 60Mun2 its one
>less problem on the genome map. Every little bit helps.
>
>Rachel.
>
Dear Rachel,
	I will contact Bill McGinnis about your query. The sequence you
sent me is identical to that of gsc, which is our PPH25, but different from
PPH13. So Bill may have confused his two genes although it is true that the
two 3' ends of PPH13 and gsc are only a few kbs apart. I will sort things
out with Bill and let you know about the outcome.
	Over ten years ago, one of my diploma students, Roland Rutschmann,
isolated the genomic region that included both PPH13 and PPH25 by low
stringency hybridization with the homeobox of the prd gene (PvuII-PstI
fragment; hence P(vuII-)P(stI)H(omology) 13 and 25!) and sequenced the
regions that hybridized with it, the homeoboxes of PPH13 and PPH25. While
he had sequenced the entire homeobox of PPH13 (it includes an intron after
the fifth amino acid of the homeodomain), he was unable to locate the last
14 amino acids of PPH25, i.e. of gsc, because they are encoded on a
separate exon and we had no cDNAs. Therefore, we do not know how close the
two 3' ends of PPH13 and gsc are but they are closer than 3-4 kb, which is
the distance of the portions of the two homeoboxes that we have sequenced.
With best regards,
Markus
>
Dear Rachel,
	A week ago, I have sent the following message to Bill McGinnis
(together with a copy of your message to me):
Dear Billy,
	Rachel Drysdale sent me this message yesterday. I promised to
contact you to clear things up. Your sequence mentioned above (VWFK
NRRAKWRKQ KREEQE) is identical to that of gsc, which is our PPH25, but
different from PPH13. So, if this sequence was derived from your 60Mun2
gene, then it must be the same as gsc (PPH25) and, accordingly, your 60Mun1
gene would correspond to our PPH13 gene.
	Over ten years ago, one of my diploma students, Roland Rutschmann,
isolated the genomic region that included both PPH13 and PPH25 by low
stringency hybridization with the homeobox of the prd gene (PvuII-PstI
fragment; hence P(vuII-)P(stI)H(omology) 13 and 25!) and sequenced the
regions that hybridized with it, the homeoboxes of PPH13 and PPH25. While
he had sequenced the entire homeobox of PPH13 (it includes an intron after
the fifth amino acid of the homeodomain), he was unable to locate the last
14 amino acids of PPH25, i.e. of gsc, because they are encoded on a
separate exon and we had no cDNAs. Therefore, we do not know how close the
two 3' ends of PPH13 and gsc are but they are closer than 3-4 kb, which is
the distance of the portions of the two homeoboxes that we have sequenced.
We know for sure that the two homeoboxes are oriented in opposite
directions with their 3' ends towards each other. Thus, the direction of
transcription of the proximal PPH25 gene is oriented towards the telomere
whereas that of the distal PPH13 gene is oriented towards the centromere.
	It seems clear to me that we are looking at the same genes, i.e.,
that PPH13 and PPH25 correspond to 60Mun1 and 60Mun2, because PPH25 is
identical to gsc and one of your genes (which one?) while the other, PPH13
(which one of yours?), is located at the same distance from the 3' end of
gsc although we seem to have a discrepancy in its orientation.
	The homeodomain sequence of PPH13 is:
QRRYR TTFNTLQLQELERAFQRTHYPDVFFREELAVRIDLTEARVQVWFQNRRAKWRNEE
with the space indicating the location of a 121 bp intron (after position
14 of the homeobox sequence). If you have sequenced both Mun1 and Mun2, it
should be easy to verify which corresponds to PPH13 and which to gsc.
Thank you very much for taking the time to look into that.
With best regards,
Markus
Bill's answer:
Subject: pph13
Status:
Markus,
Hi, I went back and looked over the sequence information we have on the
21C homeobox genes.
It seems nearly sure that PPH13 is identical to what we called 60Mun1 in
Dessain and McGinnis in 'Advances in Developmental
Biochemistry' Vol. 2, pp. 1-55. The aa sequence that you sent matches
60Mun1 at every position from +6 of your sequence to beyond the WFQNRR
motif so there seems little doubt. And we derived our sequence completely
from genomic DNA so the presence of an intron near the 5' end would explain
why the aa sequences diverge at that point. The homeobox sequences (i.e.
entire 60Mun1 and the C terminal half of the 60Mun2 homeobox), mapped about
6 kb apart on Dessain's phage map.
.
60Mun2 is identical to goosecoid/PPH25, in fact Desplan et al used the
60Mun2 clone that we sent them to isolate the gene.
Hope this is helpful to you,
Bill
	In summary, it is clear that gsc is identical to our PPH25 and to
Bill's 60Mun2 gene, which is the proximal gene of the pair of neighboring
homeobox genes at 21C8/D1, and our PPH13 is identical to Bill's 60Mun1 (not
60Mun2!) gene. The two genes are convergently transcribed, i.e. the distal
PPH13 gene from left to right, the proximal gsc from right to left. Their
3' ends are separated by less than about 4 kb. Bill's answer is clearly
not consistent with what you wrote they have published in their paper in
'Advances in Developmental Biochemistry' Vol. 2, pp. 1-55. Since our
library does not carry this journal, I could not check myself what they
wrote on page 26.
.
	If you like, you can submit our sequence of the PPH13 homeodomain
as personal communication to FlyBase. Would you like to obtain the genomic
DNA sequence of it (including the 121 bp intron after position 14)?
With best regards,
Markus