Dear Dr. Thomas,
I am curating your roughex paper for FlyBase (Thomas et al., 1994,
Cell 77(7): 1003--1014). In recording your rux6 allele I see that
you have the cytology down as being a T(2;3), but as rux is on the X
there must be a typo somewhere! Please could you let me know what the
aberration really is (and if you have any more info on the cytology of
rux6 and rux7 that would be nice too).
Many thanks for your help,
yours sincerely,
Rachel Drysdale
FlyBase (UK)
Subject: roughex paper
Priority: normal
Dear Rachel,
I am writing concerning your fax of 1 June about the paper Thomas et.
al. (1994) Cell 77: 1003.
You are indeed correct about the cytology of the allele rux6. I have
not done the cytology very well, but it appears to be a T(1;2). The
last band I can definitively see on the X chromosome is 4C1,2. The
break in 2R appears to be in 56D.
Regarding rux7, this allele is an inverted duplication of several
bands from 5D back into the rux locus. Molecularly, one insertion
breakpoint is in the rux gene, while the second insertion breakpoint is
in an adjacent transcript that is next to rux, about 5 kb away. The
insertion appears to be composed of the bands 5D3,6 (two doublets). I
know that it is an inverted duplication because the insertion loopout
is seen in hemizygous males.
I hope that this is helpful. Please contact me if you require any
additional information.
Sincerely yours,
Barbara Thomas
Subject: Re: roughex paper
Hi Barbara,
Thank you for your email. In fact I do have a couple more questions,
and I'm sure you are the right person to answer them!
What you say about rux6 suggests to me that it is a 2 break event,
with one break between 4B6 and 4C1 and the other break in 56D.
We have a statement in FlyBase that rux is
'Placed in 5C5-D6, possibly in 5D2-6 (Ashburner).'
So
either the X chromosomal break in rux6 is not related to the T(1;2)
or
the T(1;2) has more than two breaks
or
we (and Lindsley and Zimm, where this statement came from) have the
cytology of rux all wrong.
Can you say which of these three is the answer? It matters because of
the way we link aberrations to alleles. If the aberration causes the
allele then the allele name becomes part of the aberration name, and we
link allele and aberration in the database, but not if not.
>Regarding rux7, this allele is an inverted duplication of several
>bands from 5D back into the rux locus. Molecularly, one insertion
>breakpoint is in the rux gene, while the second insertion breakpoint
>is in an adjacent transcript that is next to rux, about 5 kb away.
>The insertion appears to be composed of the bands 5D3,6 (two
>doublets).
So, to check my understanding, are you saying that there is a deletion
of (not much) more than 5kb, that breaks in the rux gene and the
adjacent gene, and that the insertion of 5D3,6 occurs between the two
deletion breaks? Can you say what the cytological location of rux is?
You imply that its location is distinct from 5D3,6 when you say 'bands
from 5D back into the rux locus'.
I hope my questions aren't a nuisance. We like to get as much genetic
data from the papers as possible, to do justice to the work. The info
in this correspondance between us will be incorporated as a personal
communication to FlyBase from you, and will allow me to curate your
Cell paper more completely.
Many thanks for your help
Rachel
\--------------------------------------------------------------
Rachel Drysdale.
FlyBase (Cambridge),
\--------------------------------------------------------------
Subject: rux paper
Priority: normal
Dear Rachel,
What you say about rux6 is true, that the X chromosome breakpoint is
nowhere near the cytological position of the gene. In fact, the locus
is even more precisely localized by another deficiency that was
generated by Roger Karess in his spaghetti squash paper, Df(1)sqh,
which uncovers rux and goes from 5D1,2; 5E. This, as you've probably
worked out, puts rux in the interval 5D1,2; 5D5,6.
There is a caveat to the information that I gave you about these
alleles: I am far from being an expert at doing and scoring chromosome
squashes. However, I would be surprised if I were that far off in
reading the bands. However, I did do some analysis of rux6 at the
molecular level, and the Southerns were consistent with there being a
break in the rux gene. I guess the way that I would interpret the
results is that was a fairly large deletion associated with the
translocation that removed the DNA between 4C1 and 5D1,2; 5D5,6. I
guess the other way to figure this out would be to look at the other
translocation chromosome, which I have not done in detail.
Regarding rux7, most of what I know is from the molecular analysis - I
have not looked very hard at squashes, except to determine that there
was extra material in the 5D region and that it was probably an
inverted duplication because it was present in males. What I know from
the molecular analysis is that there indeed appear to be a deletion
between the two breakpoints: this was based on looking at three
adjacent genomic fragments (they are shown in the paper) of 3.3, 1.2,
and 6.0 kb. The 1.2 kb band, which is in the middle, is missing, and
one break is in the 3.3 kb fragment (which contains the adjacent cDNA)
and one is in the 6.0 kb fragment (which contains rux). The rux gene
resides closer in the 6 kb fragment to the 1.2 kb fragment than the
other side, so I am estimating that the amount of DNA removed is
approximately 5 kb, although it might be greater.
The sequences actually contained within the duplication come from near,
but not in, the rux locus. The rux genomic region is contained within
two overlapping cosmids: the cosmids are each about 35 kb, and the
region of overlap is about 25 kb. Rux lies almost precisely in the
middle of this 25 kb overlap. In situs to the rux7 chromosome with
the cosmids shows that one cosmid hybridizes to a single band just
distal to the insertion, while the other cosmid hybridizes both to this
band and to the insertion itself. In addition, a probe from the most
proximal end of this second cosmid also recognizes the insertion. This
probe is about 20 kb away from the 6.0 kb fragment containing the rux
gene. Based on these hybridization patterns, I am assuming that the
DNA in the insertion comes from proximal to the rux locus, and the
bands in the insertion look like they could be the 5D3,4 and 5D5,6
doublets.
I hope that this all makes sense.
With best regards,
Barbara Thomas