Subject: Re: Help FlyBase please
Dear Michael,
I am sorry for the delay, but I was away.
We just finished a manuscript regarding to the 70CD region. I think the best
to answer to copy the appropriate sections following your questions.
>
>FB knows about Hsc70C-1 at 70C. Is 'hsc70Cb' a second Hsc70 in this region ?
Yes.
>
>Do I deduce from this that:
>l(3)02402, l(3)s4868, l(3)084807 are alleles of the same gene ? If so
> what should that gene.s name be ?
I think it should be l(3)70Da.
'l(3)70Da may code for a helicase: All known mutations in the l(3)70Da gene
(cytological location: 70D1) are only semi-lethals; they all give adult
escapers, even over deficiencies, suggesting that this gene plays an almost
dispensable role in development (A. T. C. Carpenter, pers. comm.). Moreover,
homozygous third instar larvae show necrosis in salivary gland cells.
Plasmid rescue fragment sequences show that the insertion in l(3)S084807 is
15 bp distal to the insertion site of l(3)02402 and 107 bp distal to
l(3)s4868, which has been sequenced by the BDGP. A transcribed region, which
may correspond to l(3)70Da, was identified by Klämbt, Glazer and Shilo
(1992) less than 1 kb proximal to these insertions (see Fig. 3). Moreover,
sequences distal to the plasmid rescue site of l(3)02402 (see Fig. 3) reveal
an ORF of 135 amino acids, which displays low but significant similarities
to the amino-terminal region of the yeast helicase RAD26 (Acc. no. L26910).'
>
>l(3)027714, l(3)j2E11, are alleles of the same gene ? Is 027714 = S027714 ?
yes, 027714 = l(3)S027714
'l(3)S027714: The l(3)S027714 insertion is L1 lethal. The sequences adjacent
to its insertion site display similarities to those recovered from the 3'
end of the lethal insertion l(3)j2E11 (Bier et al. 1989) and to the ESTs
GH18550, GH21710 and GH21824, which all start 396 bp downstream of the
l(3)S027714 insertion point. However, l(3)S027714 complements l(3)j2E11. The
combined sequences indicate an ORF of 180+ amino acids, beginning at bp 454
and still open at the 3' end. BLASTX searches show that the putative gene
product of l(3)S027714 is highly similar to Xenopus XPMC2 (Acc. No. U10185),
a cell cycle gene that can complement mitotic catastrophe mutations in yeast
(Su and Maller 1995), and to a hypothetical exonuclease YOL080c of
Saccharomyces cerevisiae (Acc. no. Z74822).
According to the Southern blots using the plasmid rescue fragment as a
probe, the insertion on the l(3)S027714 chromosome is located between the
l(3)70Da and l(3)70Ca/b loci. However, this result is in conflict with 1)
the complementation of its lethal mutation with Df(3L)D-5rv6 (Fig. 1), 2)
the position of the l(3)j2E11 insertion in 70C5-6 (BDGP), and 3) Southern
blot experiments using genomic DNA (data not shown). It is, therefore,
likely that the P-element insertion site and lethality do not overlap. We
must consider the possibility that the Southern blot experiments were
complicated by unrecognized cross-hybridizations and, therefore, we regard
the molecular position of l(3)S027714 as uncertain.'
>
>l(3)134217, l(3)00082, l(3)07621 are all alleles of l(3)70Ca ?
>Is 134217 = S134217 ?
yes, 134217 = l(3)S134217
Here we have a complex situation. The P insertion mutants belonging to 70Ca
and 70Cb complementation groups are not complementing with the members of
the other groups, but the molecular data suggest, that they are disrupting
the same transcriptional unite
'A novel hsp70 homologue: We determined the full sequence of the plasmid
rescue clone deriving from l(3)S148513 (Acc. no. AJ132829). It measures 7728
bp and comprises the complete coding region of a novel hsp gene, which is
located proximal to l(3)S148513 insertion site (Fig. 5). The sequence data
indicate that, although we observed full complementation between mutations
in the l(3)70Ca (Carpenter 1994) and l(3)70Cb (Karpen and Spradling 1992;
Carpenter 1994) loci, these sites very likely affect the same coding region.
The l(3)70Cb allele l(3)S134217 carries a P element inserted 1246 bp
proximal to l(3)S148513, a mutation that fails to complement l(3)70Ca1
(Carpenter 1994). l(3)S148513 is likely inserted in the 5' control region of
this gene, while all three known P elements defining the l(3)70Cb
complementation group are located in the second intron: l(3)S134217 is
inserted at bp1246, just 3 bp proximal to l(3)00082 (STS Dm0089), and 276 bp
distal to l(3)07621 (bp 1522) (see Fig. 5). l(3)S134217 and l(3)S148513
exhibit similar lacZ staining patterns (Table 4), consistent with their
transgenes being driven by the same enhancer. It is conceivable that either
trans-splicing of the nascent mRNA molecules or the differential use of
promoters is responsible for this rather surprising pattern of
complementation. However, from our data we cannot exclude the possibility
that l(3)S148513 is a non-lethal insertion that carries an additional
mutation responsible for its lethality over l(3)70Ca1. At the 3' end (bp
5523 to 5846), the plasmid rescue clone overlaps with the distal end of the
P1 phage DS01408, which gave rise to STS Dm2566. This confirms the
localization and the orientation of this gene independent of the Southern
blotting experiments (Fig. 2).
Similarity searches show that this sequence is for long stretches almost
identical to the EST defined by the clot 1489 (BDGP, unpublished). The P
element of l(3)S148513 is inserted 47 bp upstream of the 5' end of the EST
clot 1489 consensus, and 15 bp in front of the longest EST (GH12450). Since
the ESTs correspond to cDNA, the exon-intron structure of the gene can be
determined. Within the sequenced part, five introns of 341 bp (bp 89/114 to
454/457), 1316 bp (bp 728 to bp 2050), 64 bp (bp 3003 to 3066), 1573 bp (bp
3598 to 5169) and 88 bp (bp 5370 to 5457) were identified. The first exon
does not code for any amino acids, but there are two alternative splice
sites for the first intron.
The coding region comprises 2412 bp that can be translated into 804 amino
acids. It starts in the second exon at bp 621 and stops at bp 6078. There is
a polyadenylation site present at bp 6241. The resulting polypeptide has a
deduced molecular mass of 88.6 kDa. Database searches using BLASTX or
TBLASTN show that this ORF displays strong similarities to members of the
mammalian HSP70/110 family. We therefore named the new hsp gene tentatively
hsc70Cb (heatshock protein cognate 70Cb). The highest similarity score was
obtained with the rat ischemia responsive 94 kDa protein (Acc. no.
AF077354). The l(3)70Ca/b sequence is not particularly related to the HSP70
cognate hsc70A1 (Rubin et al. 1993), which is also located in 70C (Craig,
Ingolia and Manseau 1983); it is therefore very likely that there are at
least two HSP70 homologues in this region. Both l(3)S148513 and l(3)S134217
are embryonic lethal as homozygotes, indicating an essential role of this
gene in Drosophila development. A universal role of the gene product in the
living animal is also supported by the observation that the ESTs derive from
cDNA libraries of different stages and tissues.'
>
>What is the relationship between l(3)70Ca and 'hsc70Cb' and or Hsc70C-1 ?
I think, the text above answers this, l(3)70Ca identifies 'hsc70Cb', and the
sequence of hsc70Cb is different to Hsc70C-1.
>
I admit, the whole picture is complex. But this is what we have.
With the best regards
Peter
Peter Maroy, Deptartment of Genetics and Molecular Biology
A.Jozsef University, Kozepfasor 52., H-6726 Szeged, Hungary