See the embryonic expression pattern of hrg at the Berkeley Drosophila Genome Project Patterns of Gene Expression Site.
The expression profile of PAP during Drosophila development was assayed using a polyclonal antibody. During oogenesis, PAP is detected in both the nucleus and cytoplasm of nurse cells and follicle cells. PAP is also present at a low level in oocyte nucleus and cytoplasm. PAP was overexpressed in the female germline using a UASp-hrg transgene under the control of the female germline-specific driver nanos-Gal4:VP16 (nos- Gal4). In UASp-hrg; nos-Gal4 females, PAP accumulates to a high level in nuclei of nurse cells and oocyte and to a lesser extent in oocyte cytoplasm. Maternally provided PAP is detected in just laid embryos where the protein is distributed uniformly. During embryogenesis, the amount of PAP increases until cellularized blastoderm stage and remains stable during gastrulation. The subcellular distribution of PAP was analysed in cellularized blastoderm embryos. PAP accumulates in nuclei and is present at a lower level in the cytoplasm, as was reported for PAP II in human somatic cells (Schul, 1998; Kyriakopoulou, 2001). A high level of PAP accumulates in early embryos coming from females where PAP is overexpressed in the germline. In contrast, PAP is not detected in hrgPAP12 mutant embryos that show no hrg early zygotic transcription. This shows that the antibody is specific for PAP. A major protein is detected in Drosophila extracts with this antibody by Western blot. This protein has a mol. wt of 75 kDa, which is the expected molecular weight for Drosophila PAP. Its level increases during the first hour of embryogenesis; it is very abundant in embryos from females overexpressing PAP in the germline and in late embryos overexpressing PAP ubiquitously, and is absent in hrgPAP12 mutant larvae (Juge, 2002).
These data show that hrg encodes a single form of PAP. This protein is mostly nuclear in somatic cells and is present in the cytoplasm of oocytes and early embryos where cytoplasmic polyadenylation takes place (Juge, 2002).
Since mutation at the hrg locus results in loss of the normal wing margin, an examination was carried out to see whether expression of the ct and wg genes might be altered by mutations in the hrg gene, namely, by hrgP1 and hrg10 (Murata, 1996b). Reduced expression of lacZ directed by the ct margin enhancer along the boundary of the dorsal/ventral (D/V) compartment was evident around the anterior/posterior (A/P) boundary and, for the most part, in both the anterior and posterior regions of the wing pouch. Reduced expression of wg was observed specifically at the wing margin, while expression of wg in other regions of the wing disc, as well as in other imaginal discs such as the leg discs, was unaffected by the mutations in the hrg gene. Thus, it appears that hrg might act specifically in the regulation of expression of the wg gene at the presumptive wing margin rather than in the regulation of the overall expression of wg. The reduced levels of expression of ct and wg that are associated with mutations in the hrg gene suggest that loss of expression of these genes might be responsible for the loss of the normal wing-margin structure in the hrg mutants (Murata, 2001).
Since the duplication Dp(2;Y)53D;57C, which covers the hrg locus (Murata, 1996), rescues the notched phenotype of adult wings of hrgP1 mutant flies, fragments of genomic DNA were sought that encode genes that could restore hrg activity. Among the DNA fragments isolated from a wild-type genomic DNA library, a 6.8-kb SalI fragment (hrg72) was identified that was able to rescue the adult wing phenotype when introduced into hrgP1 and hrg10 mutants by P element-mediated transformation. DNA sequence analysis of phrg72 and embryonic cDNA clones identified the hrgE transcript (embryonic hrg) as a transcript that corresponds to part of the genomic DNA of phrg72. Northern blots of larval RNA with a cDNA probe for hrgE and the phrg72 probe demonstrated that a 4.0-kb transcript is the major hybridizing species in wild-type larvae and that the level of this transcript is eightfold less in hrgP1 and threefold less in hrg10 mutants, respectively. Neither hrgP1 nor hrg10 are null alleles of the hrg gene because RT-PCR detects coding regions of hrg in these alleles (Murata, 2001).
Using the Gal4 line C-765, it was shown that introduction of the UAS-hrgE transgene rescues the wing phenotype in hrg10 mutant flies. Thus, hrgE is sufficient to reverse the hrg mutation. Taken together, the results suggest that the level of expression of hrg is reduced in flies with the mutant alleles of the hrg gene and that the reduced expression of hrg results in failure to develop a normal wing (Murata, 2001).
The hrg gene appears to function at the wing margin both correctly and specifically since no defects other than abnormal wing margins are found in hrgP1 and hrg10 flies (Murata, 1996). The expression of hrg is detected in most tissues, including the imaginal discs, fat bodies, salivary glands, and muscles of wild-type larvae. Such ubiquitous expression of hrg is impaired in larvae with mutant hrg alleles (Murata, 2001).
To examine whether a defect in PAP might be responsible for the wing phenotype of hrg mutants, cDNA for bovine PAPII was introduced into flies by P element-mediated transformation. All hrg10 flies with the Gal4 line MS1096 and UAS-bPAPII had wild-type wings. The wing phenotype of the hrg10 mutants did not return to the wild-type wing phenotype when hrgE that encodes not aspartic acid, but alanine at position 175, was expressed in the hrg10 mutants by introduction of UAS-hrgD175A and the C-765 Gal4 line. These results suggest that the wing phenotype of hrg might be due to reduction of the expression of a gene that encodes a protein that is functionally similar to PAP (Murata, 2001).
The hrg gene was localized by in situ hybridization to position 56E5- 6 on chromosome II. A collection of 21 P{lacW}-induced lethal mutants on chromosome II, containing the P insertion in the vicinity of region 56E5- 6, were screened by Southern hybridization. In three of these stocks, l(2)k07618, l(2)k07609 and l(2)k07626, the P element was found to be inserted in the 5'-untranslated region (UTR) of hrg. DNA from these three stocks shows the same restriction profile, suggesting that it contains the same P-element insertion. The insertion was mapped in the l(2)k07618 stock and is located 261 bp downstream of the hrg 5'-most transcription start site. Although this insertion was isolated in a P-induced lethal mutant collection, it does not cause lethality. The mutation inducing lethality in the l(2)k07618 stock was removed by recombination. This stock was named hrgPAP2. hrgPAP2 is viable and fertile, although ~50% of homozygous hrgPAP2 individuals die as first instar larvae. New hrg mutants were generated by imprecise excision of the P-element in hrgPAP2. Three homozygous lethal mutants, hrgPAP45, hrgPAP21 and hrgPAP12, that show a deletion in the coding sequence were used in further studies. The first six residues and the first 44 residues are deleted in hrgPAP45 and in hrgPAP21, respectively. In hrgPAP12, more than the N-terminal third of PAP (243 residues), including the catalytic core, is deleted. In all three mutants, most of the 5'-UTR of the longest mRNAs as well as the embryonic transcription start site are missing. The three mutants are lethal from late embryonic to second instar larval stages, with hrgPAP45 showing the weakest phenotype and hrgPAP12 the strongest. They do not complement each other and are, therefore, alleles of the same gene. Late embryos of all three mutants show no strong phenotype; however, they present a slight defect in head skeleton (distortion of the dorsal bridge). Lethality of hrgPAP45 and hrgPAP21 is rescued with a hrg genomic transgene. However, only 20% for hrgPAP45 and 5% for hrgPAP21 of the expected rescued progeny survive to adulthood, suggesting that hrg in the transgene is not fully expressed. Lethality of the strongest allele hrgPAP12 is not rescued with the genomic transgene, but is rescued with the transgene UASp-hrg expressed ubiquitously with the driver daughterless-Gal4 (da-Gal4). It as verified that hrg mutants described earlier (Murata, 2001) are alleles of the gene described in this study. hrgP1 is not lethal but shows a notched wing phenotype. hrgPAP12 does not complement hrgP1, since hrgP1/hgPAP12 adults have a pronounced notched wing phenotype (Juge, 2002).
Taken together, these data demonstrate that strong alleles of the hrg gene have been induced that encode Drosophila PAP and that the lack of PAP in Drosophila is lethal (Juge, 2002).
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date revised: 17 January 2002
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