Subject: Helping FlyBase: Tctex
Dear Dr. Caggese,
We have been curating the DNA sequence records for FlyBase and have a
question for you. You have recently submitted a sequence record,
accession number AF123058; AAD30033 for the Tctex gene, which you say
encodes a dynein light chain and maps to 90F9--91A2. We already have a
record for a dynein light chain gene at 90F, and I was wondering
whether you knew if this was the same gene as yours? I enclose what we
have about the other gene below, for you to see. I will be querying
the authors of the abstract to see if they can add anything more.
\*a Dlc90F
\*z FBgn0024432
\*c 90F
\*c Left limit from (method unavailable) (FBrf0101355)
\*c Right limit from (method unavailable) (FBrf0101355)
\*x FBrf0101355 == Li et al., 1998, A. Conf. Dros. Res. 39: 318A
\*e Dynein light chain 90F
\*c 90F
\*d dynein light chain
\*p Lethal lines have been obtained by imprecise excision of a P-element
\*p inserted 800bp upstream of the transcription start site of Dlc90F.
\*u Southern blot analysis indicates that Dlc90F is encoded by a single
\*u copy gene and is expressed as a single transcript throughout Drosophila
\*u development. Western blot experiments show that the 14kD light chain is
\*u co-purified with cytoplasmic dynein from embryos and there exist two
\*u isoforms, suggesting a post-translational regulation of the light chain.
Also, you annotated your sequence with three intriguing gene symbols
> gene 1672..2476
> /note='EP(3)3634, l(3)04091, l(3)05089'
This implies to me that you know that the flanking sequence of
insertions in genome project lines EP(3)3634, l(3)04091 and l(3)05089
matches sequence corresponding to Tctex. This is of relevance because
we (FlyBase) already have a gene entry for l(3)04091, with an allele
l(3)0409104091. If that maps to Tctex I should merge the records
for l(3)04091 and Tctex (keeping Tctex as the valid symbol) and record
an allele Tctex04091 to correspond to what we have now as
l(3)0409104091. Similarly, if the P insertion in line l(3)05089 maps
to the Tctex gene, I need to record an allele Tctex05089 (we already
have and allele of quag, quag05089, at 94A8--B5, but we know that the
quag phenotype in the line is separable from the P insertion). Finally, if
EP(3)3634 has an insertion in/affecting the Tctex transcription unit I
would record it as an an allele of Tctex, Tctex3634.
So, if you could give me this little bit more information, we would be
most grateful.
With best wishes,
Rachel.
Subject: Re: Helping FlyBase: Tctex
Dear Rachel,
I isolated a cDNA encoding the putative Drosophila tctex-1 gene at 91F9-11
by a strategy for identification of nuclear genes encoding mitochondrial
protein (accession number Y08968; Caggese et al.(1999). Mol. Gen. Genet.
261: 64-70).
Developmental Northern blot analysis reveals a 0.85-kb transcript in all
stages examined. This transcript is also present at high levels in the
oocyte nurse cells.
Some overlapping genomic clones were isolated and a subclone of 3115
nucleotides that contained the entire tctex-1 gene was sequenced (accession
number AF123058). Sequence analysis revealed that no introns interrupt the
tctex-1 cDNA sequence .
A search in the GenBank and Expressed Sequence Tag database using genomic
nucleotide sequences revealed that two unidentified ESTs (LD12970,
accession numbers AA438652; CK01205, accession number AA141098) are also
transcribed from the genomic subclone.
EST LD12970 (clot 3142): 1262...540 exon; 539...1.. intron
tctex: 1672...2176
EST CK01205: ..3115...2662
(the numbers correspond to positions of the genomic subclone)
I received many available single P-element insertion mutants mapping at
90F-91A from Bloomington, Berkeley and Szeged stock collections. In order
to identify mutant alleles of the tctex-1 gene we used PCR between
divergent primers derived from the tctex-1 nucleotide sequence and a primer
derived from the 31-bp terminal inverted repeat sequence of the P element.
A specific amplification product was detected in the genomic DNA from:
l(3)05822 single P-element insertion mutant mapping at 90F9-10. Sequence
analysis revealed that a PZ element is present in this mutant stock at
position 756-757 of the genomic clone and disrupts the EST sequence LD12970.
EP(3)3634 single P-element insertion mutant mapping at 91F9-11;91A1.
Sequence analysis revealed that a EP element is present in this mutant
stock at position 1671-1672 of the genomic clone and disrupts the tctex-1
transcription unit.
l(3)04091 single P-element insertion mutant mapping at 91F9-11; 91A1.
Sequence analysis revealed that a PZ element is present in this mutant
stock at position 1710-1711 of the genomic clone and disrupts the tctex-1
transcription unit.
l(3)05089 single P-element insertion mutant mapping at 91F9-11; 91A1.
Sequence analysis revealed that a PZ element is present in this mutant
stock at position 1714-1715 of the genomic clone and disrupts the tctex-1
transcription unit.
BLAST analysis of tctex-1 cDNA sequence showed that 35 nucleotides in the
UTR are identical to the 35 nucleotides 5' flanking sequence of P element
in the ms(3)05090 (sdl-2, seedless; Castrillon et al. (1993) Genetics
 135:489-505 ) reported as single insertion line at 84F1-84F16 region.
In reality, sdl-2 is a allele of tctex-1 at 91F9-11; 91A1 since specific
amplification products were detected in the genomic DNA of sdl-2 mutant
flies by using different primers derived from the tctex-1 nucleotide
sequence and a primer derived from the 31-bp terminal inverted repeat
sequence of the P element. Sequence analysis confirmed that in this mutant
allele a P element disrupts the dtctex-1 transcription unit by insertion at
positions 1721-22 of the genomic clone.
I don't know if multiple inserts are present in this line, but a P element
is certainly inserted in the tctex-1 gene at nucleotide position which is
reported in Berkeley Fly Database.
The phenotypic analysis of the EP(3)3634, l(3)04091, l(3)05089 and sdl-2
(ms(3)05090) mutations showed that tctex-1 is required for production of
functional sperms.
The manuscript entitled 'The Drosophila melanogaster homolog of tctex-1, a
putative murine t-complex distorter encoding a dynein light chain, is
required for production of functional sperm' will be submitted for
publication as soon as possible.
With best wishes,
Corrado
Prof. Corrado Caggese
Istituto di Genetica
Universita di Bari
via Amendola 165/A
70126 Bari, Italia